Along with the development of science and technology, lanthanide‐doped upconversion nanostructures as a new type of materials have taken their place in the field of nanomaterials. Upconversion luminescence is a nonlinear optical phenomenon, which absorbs two or more photons and emits one photon. Compared with traditional luminescence materials, upconversion nanostructures have many advantages, such as weak background interference, long lifetime, low excitation energy, and strong tissue penetration. These interesting nanostructures can be applied in anticounterfeit, solar cell, detection, bioimaging, therapy, and so on. This review is focused on the current advances in lanthanide‐doped upconversion nanostructures, covering not only basic luminescence mechanism, synthesis, and modification methods but also the design and fabrication of upconversion nanostructures, like core–shell nanoparticles or nanocomposites. At last, this review emphasizes the application of upconversion nanostructure in detection and bioimaging and therapy. Learning more about the advances of upconversion nanostructures can help us better exploit their excellent performance and use them in practice. Through adjusting the relative intensity of these particular emission bands, the emission from green to red can be obtained. The two characteristic emission bands of Tm3+ are respective about 800 and 480 nm, and blue light was apparent because the light of 800 nm is not visible. Ho3+ possesses similar characteristic emission bands to those of Er3+. As another kind of dopants, sensitizers are responsible for the absorption of energy from excitation source and then transferring it to activators. Yb3+ and Nd3+ are the most commonly used sensitizers.3+ and Nd3+ not only has large absorption cross‐section at 980 and 808 nm, respectively, but also could be in resonance with activators. In addition to dopant, host matrix provides a platform for energy transfer.Because of the different luminescence mechanism of upconversion materials against the traditional fluorescent ones, many research teams were committed to the study of their luminescence mechanism, which leads to a developing understanding of their luminescence mechanism. In most cases, upconversion luminescence (UCL) was derived from more than one fundamental mechanism. Among the numerous fundamental mechanisms, excited state absorption (ESA), energy transfer upconversion (ETU) are the most significantly influential factor. Dopants and matrices are the essential elements for upconversion. Activators act as dopant, to absorb and then release the energy in the form of fluorescence. The common activators for upconversion are ErSynthesis plays a critical role in determining the structure, composition and properties of resulting materials. The resulting upconversion nanostructures may be either hydrophobic or hydrophilic, which have their merits and demerits, though the former surface feature is more common. For example, the upconversion nanostructures with controllable shape and size can be prepared by thermal decomposition, high temperature co‐precipitation and solvothermal method.Utilizing newly developed materials for particular applications is the ultimate goal of scientific research, which applies to the upconversion fluorescent nanomaterials. Given low background noise and excitation located in the IR region, lanthanide‐doped upconversion nanostructures can be used in many aspects, such as physics, chemistry, biology, and medicine, especially, as a probe for detection, bioimaging, and therapy.This review aims to discuss the existing advances in the reasonable design and synthesis of lanthanide‐doped upconversion nanostructures. To begin with, we attempt to introduce the mechanism of lanthanide‐doped upconversion nanostructures from host matrices to dopants in Section 2UCL is an anti‐Stokes optical phenomenon. Simple upconversion nanostructures normally contain dopants and host matrices that are the key factors determining luminescence efficiency. Dopants, including sensitizers and activators, provide a luminescence center, while host matrices supply a platform for energy transfer between the dopants and drive them into optimal position.2.1n5s25p6 (n varies from 0 to 14) electronic configuration. The partially filled 4f electronic shell that is critically relevant to photoluminescence is protected by outer 5s and 5p electronic shells from external environmental disturbances.n between 0 and 14 elicits lanthanide ions energy‐enriched levels, which contributes greatly to broadband spectrum. Upconversion is a nonlinear optical phenomenon, whose fundamental mechanisms consist of ESA, ETU, photon avalanche (PA), cooperative energy transfer, and cross‐relaxation. Of them, ESA and ETU are responsible for UCL efficiency.It is well known that lanthanide ions exhibit a 4f Figure3+, Tm3+, and Ho3+, commonly play an activating role in single‐doped systems, which can be explained by ESA. For single‐doped systems, Er3+ has a relatively high quantum yield given the high similarity of ≈980 nm in the energy gaps between 4I11/2 and 4I15/2 states and between 4I11/2 and 4F7/2  generated by 3H4–3H6 transition, which is favorable for biological tissues because of the deep tissue penetration and low heat effect. Three additional primary upconversion emission bands stay around 350, 450, and 479 nm corresponding to the transitions 1D2–3H6, 1D2–3F4, and 1G4–3H6, respectively. In terms of Ho3+ ions, there are two main upconversion emission bands, including the red centered at 650 nm originated from the transition 5F5–5I8, and the green centered at 540 nm originated from the transitions 5S2/5F4–5I8. Low doping concentration is common in single‐doped system (less than 3% Er3+ and no more than 1% Tm3+) to avoid concentration fluorescence quenching that can be incurred by increase in harmful nonradiative transitions derived from high doping concentration.ESA, normally doped with low Lanthanide ion concentration <1%), is responsible for the single ion based process. As depicted in %, is resUnlike ESA based on single ions, ETU always occurs in two neighboring ions regardless of their chemical nature.3+ ions with a simple energy level structure are typical sensitizer, given to their large absorbing cross‐section about 980 nm corresponding to 2F7/2–2F5/2 transition, and matched energy well with a large number of f–f transitions of the typical activators . The doping concentration of Yb3+ ions should be controlled at a medium level (20%–40%) to eliminate the hazardous concentration quenching. Selection of various dopants or doping concentrations will lead to diverse colors of upconversion emissions. Liu and co‐workers utilized Yb3+/Er3+ and Yb3+/Tm3+ co‐doped NaYF4 NCs to yield a series of colors of light.3+, Nd3+ sensitizer also has increasingly been sought. Compared to Yb3+, the thermal effect of Nd3+ is lower, and more importantly, unlike the centered at 980 nm absorption peak of Yb3+, the absorption peak of Nd3+ does not overlap the absorption peak of water, which is more suitable for biological applications. In some cases, Nd3+ ions and Yb3+ ions act jointly as sensitizers.3+ ions are excited metastable level 4F5/2 from ground level 4I9/2 by absorbing a pump photon, reaches energy level 4F3/2 by nonradiation transition, and then transfers the energy to Yb3+ ion, and finally, Yb3+ ion transfers the energy to activator. However, for the sake of improvement in UCL efficiency and feasibility for further applications, the upconversion systems doped with two sensitizers will have a complicated reaction mechanism, like core/shell structures,Dopant ions can be categorized into activators and sensitizers. As Figure 2.22+,2+,2+,2+,2+,4+,4,4,4,4,5,5,UCL efficiency is closely related to host matrices, given their critical role in determining the surrounding environments of dopant ions, such as spatial distance, coordination numbers, and energy transfer efficiency. Selection of proper host materials is of paramount importance in which a few basic requirements are highly demanded, including optical stability and resembling ionic size to that of the dopant ions. Thus, the inorganic compounds containing alkaline earth ions, for instance Ca4 whose UCL efficiency is substantially reinforced due to the transformation from α to β phase with a more asymmetry. Symmetry of rare earth doped host crystal field could be tailored to change the spatial distance between the luminescent centers and lead to some other energy transfer processes.In general, radiative transitions of rare earth ions are forbidden by quantum mechanical selection rules. However, such a forbidden nature can be broken by the crystal field of host matrix. When lanthanide doping ions are introduced into an asymmetrical crystal field, their 4f state shall mix with higher electronic configurations, leading to a higher degree of asymmetry of the host matrix and consequently a better UCL efficiency. A good example can be seen in lanthanide doped β‐NaYF3+ ions in NaYF4 facilitates the crystal phase transition from cubic to hexagonal.+ ions that are optically inert have been extensively used to modify the host crystal field.+ ions having the smallest cationic radius are expected to enter lattice sites or interstices randomly, which makes Li+ ions more suitable to modify the host crystal field. Zhang and co‐workers reported a significant enhancement (by 25 times) of the visible upconversion emissions in Y2O3:Yb,Er nanoparticles through Li+ doping for the first time.+ doping produces a more than 30‐fold increase in upconversion emission for NaYF4:Yb,Er upconversion nanoparticles (UCNPs)  Figure2a.48 I2F7 host material adopts an orthorhombic crystallographic structure to construct a “dopant ions spatial separation” structure at the sub‐lattice level with enhanced UCL efficiency . KYb2F7 material plays a dual role of host matrix and sensitizer, which is favorable for the generation of multiphoton upconversion.In general, concentrated luminescence centers shorten the spatial distances between luminescence centers, which leads to the development of some detrimental energy transfer and luminescence quenching effect. As such, concentrations of activators and sensitizers have to be strictly controlled to perform their desirable functions. However, a latest report claims that the use of a new class of KYb3 was adopted as host matrix to obtain pure single band upconversion .2+ ions within the host matrix and the dopants ions and a pure single‐band upconversion emission concentered on the red and NIR spectral regions was obtained. In addition to the host materials containing Mn2+, red single band can be observed in NaScF4, NaSc2F7, and YOF host materials.The color derived from upconversion emission is normally adjusted through controlling concentration or species of the doping rare earth ions. In some particular cases, the host materials, nevertheless, also have an influence on the upconversion emission color, as indicated by the latest report of Liu's group, where KMnFFurthermore, crystal grain size of host matrix is one of primary factors affecting the luminescence efficiency. For upconversion materials a smaller grain size gives rise to a lower UCL efficiency, which is attributed to the higher density of surface defects and more serious energy transfer loss associated with smaller grain size.2.3Energy transfer, a great physical concept, applies well to all upconversion process. Here, we discuss energy transfer process from three main contents, i.e., energy transfer in core@shell nanostructures, localized surface plasmon resonance (LSPR) assisted energy transfer, and luminescence resonance energy transfer (LRET).4:Yb,Er@KYF4,4:Yb,Er(Tm)@NaGdF44:Ln@CaF24:Tm@CaF24:Yb,Er@NaYF4:Yb,Tm was reported to that of NaYF4:Yb,Er,Tm.Figure 3+ ions, and accumulated by Tm3+ ions in the core area. Afterward, the energy transfer from Tm3+ ions to Gd3+ ions in the intermediate layer, and finally is captured by X3+ ions in outer shell for UCL under 980 nm irradiation. Apart from the host matrix containing Gd3+ ions, Yb3+ doped host materials also have a similar property. Zhao and co‐workers designed an Nd3+‐sensitized core–shell–shell nanostructure of NaYF4:Yb,X@NaYF4:Yb@NaNdF4:Yb. The Nd3+ ions in the outer shell was excited by 800 nm irradiation, and with an aid of Yb3+ ions, energy migrates from Nd3+ ions to X3+ ions.Shell layers in core@shell nanostructures are either active and inactive in terms of UCL.4:Yb,Tm NCs with plasmonic gold nanostructures.4:Yb,Tm NCs, emission intensities of 1D2‐3F4 and 1G4‐3H6 transitions were increased more than 150%, while an increase of only ≈50% was observed in 1G4‐3F4 transition. The enhancement could be attributed to the increase in radiative decay rate and emission efficiency. On the contrary, a quenching effect may be caused by the considerable scattering of excitation irradiation, when NaYF4:Yb,Tm nanocrystals was embraced by gold shells. As Figure 3+ ions to Er3+ ions, which is enhanced at least 6 folds on fabricated gold pyramid pattern.In recent years, noble metals have been extensively studied due to their excellent optical properties, such as strong visible light absorbing and scattering. LSPR can occur between noble metals, like Ag or Au, and phosphors, when the confined free electrons of noble metals are resonant with frequencies close to those of the passing photons. In general, the enhancement effect of LSPR derives from increases in excitation and emission rate. The excitation rate is elevated by the amplification of local incident electromagnetic fields when the excitation band of upconversion nanostructure couples with the LSPR band of noble metals. The increase in emission rate, when the emission band upconversion nanostructure is resonant with the LSPR frequencies of noble metals, would not only promote radiative decay rate but also promote nonradiative decay rate, which can give rise to emission quenching. In recent years, various nanostructures of noble metals, including nanoparticles, nanowires, and nanoarrays, have been employed to investigate the effect of LSPR. Zhang et al. reported a modulation of upconversion emission through hetero‐integration of NaYF4:Yb,Er nanoheterostructures (CSNY), where the CdSe nanoparticles act as an acceptor absorbing green upconversion band and emitting red light under 980 nm excitation , referring to energy donor and acceptor, requires a certain degree of overlapping between absorption band of acceptor and emission band of donor, and a close spatial distance between donor and acceptor for energy transfer. In UCNPs based LRET nanocomposites, UCNPs are usually used as energy donor, while a number of dyes, quantum dots (QDs) or metal nanoparticles act as acceptor. UCL was absorbed by acceptor and subsequently produced new emission colors. Perepichka and co‐workers reported a novel CdSe/NaYFn Figure e,f.67 In3Due to the development of nanoscience and nanotechnology, synthesis of UCNPs with controllable size, crystalline phase and composition has gradually become mature. Synthetic UCNPs can be divided into the oil‐dispersible and water‐dispersible based on their decentralized nature. Oil‐dispersible UCNPs are produced through employing oleic acid (OA) and oleylamine (OM) as surfactant. Such hydrophobic UCNPs exhibit excellent dispersibility, uniform size distribution, high crystallinity, and superior UCL properties. However, some particular UCL related applications including bioimaging and theropies, require water‐dispersible UCNPs, which inspires studies of converting hydrophobic UCNPs into hydrophilic counterparts. In order to facilitate biological applications, direct synthesis of water‐dispersible UCNPs has been developed. Surface of water‐dispersible UCNPs is normally covered by hydrophilic polymer or molecules to achieve sufficient stability in service. In this section, we will discuss the synthesis and modification of UCNPs from three aspects: synthesis of hydrophobic UCNPs, direct synthesis of hydrophilic UCNPs, and conversion of hydrophobic UCNPs to hydrophilic UCNPs.3.1Considerable efforts have been devoted to synthesis of hydrophobic UCNPs with controllable shapes, sizes and phase, which delivers a great impact on upconversion emission efficiency and their applications.3 triangular nanoplates via decomposing La(CF3COO)3 precursors in a OA/ODE solution at high temperature.4:Yb,Er and NaYF4:Yb,Tm UCNPs by thermal decomposition of sodium and lanthanides trifluoroacetates in OM,4 and NaYF4:Yb,Er/Tm NCs with controllable size in OA/OM/ODE.4:Yb,Er UCNPs, they proposed a scheme of the nucleation stages combining transmission electron microscopy, X‐ray diffraction and upconversion emission spectroscopy.Figure4:Yb,Er from their α counterparts was depicted in Figure 4:Yb,Er precursors dissolve in the beginning period, and Ostwald‐ripening process is enhanced during the α → β phase transition, owing to the broad size distribution of the dissolved α‐NaYF4:Yb,Er. In terms of route B, addition of CF3COONa in this system suppresses Ostwald‐ripening, a nucleation process initiating at low temperature and regrowth at high temperature, which is ascribed to the high concentration of monomers. At the stage of α → β phase transition, the size increases uniformly. Murray et al. obtained highly uniform β‐NaYF4 UCNPs with a diverse family of morphologies  by adjusting the synthetic conditions, including the reaction time, the ratio of OA and ODE, and the concentration of precursors  and high crystallinity nanocrystals will be synthesized by Ostwald‐ripening. For example, Zhang et al. reported the synthesis of lanthanide‐doped pure hexagonal‐NaYF4 UCNPs via high‐temperature coprecipitation.4‐based UCNPs with different shapes could be obtained via adjusting OA and ODE  to hexagonal phase (NaScF4) would be achieved. The raw materials of rare earth chlorides could be replaced by other rare earth inorganic salts. Recently, Liu's group reported the preparation of lanthanide‐doped NaGdF4 nanoparticles by a similar process, which just uses acetates counterparts as starting materials thermal synthesis.4 (M = Rare earth).3)3 as precursors and OA as a stabilizing agent and obtained nanostructured arrays that were composed of hexagonal nanotubes with lengths of ≈500 nm and outer diameters of ≈250 nm thermal synthesis has attracted much attention for synthesizing high quality NCs with well controlled size and shape, due to its low cost. At a certain temperature and pressure, water or other solvents is at a critical or supercritical state, which leads to an elevated reaction activity. So, the physical and chemical properties of the substances in the solvents were altered greatly. The most popular procedure to prepare hydrophobic NCs is the liquid−solid‐solution (LSS) process that was proposed by Li's group Figure6a.87 ASome other synthetic methods have also been developed to obtain hydrophobic UCNPs, such as ionic liquid‐based,3.2Regarding bioapplications, UCNPs with high water solubility is desired, as such secondary modifications are essential to convert hydrophobic UCNPs into hydrophilic ones, which inspires study of direct synthesis of hydrophilic UCNPs. Direct synthesis of hydrophilic UCNPs often employs water or polyol as solvent and hydrophilic polymers and molecules as surfactant. The capping ligands of the finally obtained hydrophilic UCNPs normally contain special reactive groups and can further conjugate with biomolecules or functional groups.Figure4:Yb,Er,4:Yb/Er upconversion phosphors with carboxyl‐functionalized surfaces, to prepare hydrophilic NaYF4:Yb/Er upconversion phosphors via one‐step hydrothermal method.4:Yb/Er upconversion phosphors can be controlled. Furthermore, the carboxyl‐functionalized hydrophilic NaYF4:Yb/Er upconversion phosphors can directly conjugate antibodies for biodetection. Our group also has conducted some investigations in terms of preparation of hydrophilic lanthanide‐doped UCNPs.4 UCNPs were obtained via a fast, simple, and environ‐mentally friendly microwave‐assisted modified polyol process with PEI as surfactant.4 pure phase transition from cubic to hexagonal was achieved by modulating the ratio of Gd3+:F−. The upconversion emission from visible to near‐IR, even white light, was tuned via adjusting the doping concentrations of the rare earth luminescent centers.The above mentioned hydro(solvo)‐thermal synthesis is a typical synthetic method, which is applicable to direct synthesis of hydrophilic UCNPs.4:Yb,Er upconversion nanospheres were fabricated by using Y(OH)CO3:Yb,Er nanospheres as sacrificial templates via a surface‐protected “etching” and hydrothermal ion‐exchange process by Lin's group.3:Yb,Er precursors. After addition of NaBF4, the as‐obtained solution was transferred into a Teflon autoclave for a certain time period under a certain temperature. At high temperature and pressure, the fluoride source, i.e., NaBF4, can gradually release H+ and F− ions. The H+ ions corroded Y(OH)CO3 nanospheres and resulted in a large quantity of Y3+ ions. Na+, Y3+ and F− ions reacted to generate α‐NaYF4. PEI coated on the surface of Y(OH)CO3 can effectively protect them against rapid dissolution incurred by H+ ions, as a result, α‐NaYF4:Yb,Er upconversion nanospheres with hollow structure were obtained. Furthermore, FA was conjugated on the surface of α‐NaYF4:Yb,Er nanospheres, due to the presence of free amine groups. Using the identical synthetic process, hollow CaF2, GdVO4, and NaREF4  microspheres can be fabricated.2O3:Yb,Er hollow spheres with uniform morphology and controllable inner structure via hydrothermal method followed by temperature‐programmed calcination 4) to replace the original organic ligands of nanocrystal surface. Such strategy does not work well on other diazonium tetra‐fluoroborate compounds.4− anions attaching weakly to the surface after replacement can render NCs easily a high dispersibility in various polar, hydrophilic solvents with a marginal impact on the particle size and shape. This approach is widely applicable to a great number of NCs with varying shape and size. This method is of importance because the obtained ligand‐free NCs can be stored in solvents over a long time period without evident aggregation.When an acidic pH is applied, oleate is protonated completely and detached from the UCNPs surface. The charge repulsion on the surface stabilize UCNPs. Liu's group adopted a similar strategy to prepare bare sandwich‐structured UCNPs with short spatial distance for energy transfer.3.3.32MA‐co‐SEMA) copolymers as the new capping ligand.2 in the ligands exchange procedure, the luminescence intensity would be reinforced.Ligand exchange is an effective technique to replace the original hydrophobic ligand coating on the UCNPs surface with hydrophilic ones. The process is easy to operate, and exhibits a negligible effect on the morphology of the yielded UCNPs. The driving force behind this reaction is that hydrophilic ligand has a stronger coordination ability to lanthanide ions than original hydrophobic ligand. A variety of organic molecules or polymers, such as PAA,3.3.4Ligands interaction can be categorized into ligands layer‐by‐layer assembly and ligands attraction. This approach would bring a hydrophilic shell coating on the OA‐capped UCNPs for conversion of hydrophobic UCNPs into hydrophilic ones.2 groups could attach to biotin for further fluorescence resonant energy transfer. Li and co‐workers developed water dispersible UCNPs by self‐assembly interaction between the host molecule alpha‐cyclodextrin (α‐CD) and the guest molecules OA , PSS = poly(styrene sulfonate)).A Figure e.119 ThiA Figure f.23 In tAbove all, the modifications of converting hydrophobic UCNPs into hydrophilic ones have merits and drawbacks. Some properties of UCNPs, including morphology, monodispersity, and UCL, may be affected more or less. The perfect modifications with the control of particle size and homogeneity and the preserved UCL need to be developed.44@NaYF4 of core or some other substances whose lattice is similar to host matrices of cores, like α‐NaYF4@CaF2. As Section For the upconversion nanostructures, it is important to improve their UCL efficiency. So far, numerous efforts have devoted to improving UCL efficiency of upconversion nanostructures, such as seeking optimal matrices, tuning the doping concentration, constructing core–shell nanostructures, and so on. Core–shell nanostructures play an important role in upconversion nanomaterials, which can not only improve the optical properties but also combine discrete functional units together. Core–shell structures are generally divided into two classes: epitaxial core–shell nanostructures, and nonepitaxial ones. Shell of the epitaxial core–shell nanostructures must have low lattice mismatch with the core, which could decrease surface defects and improve UCL efficiency. The shell layer could be host matrx, like NaYF4:Yb,Tm Nanocrystals through thermal decomposition.4 nanoparticles via heating‐up method was reported by Liu et al.Heating‐up method is a commonly used synthetic method to obtain core–shell structures through seed‐mediated epitaxial growth.Figure4:Yb,Er/NaYF4 UCNPs was achieved by successive layer‐by‐layer method. However, quantum yield was 0.47 ± 0.05% for the heterogeneous doping NaGdF4:Yb,Er/NaYF4 UCNPs. The upconversion emission of NaGdF4:Yb,Tm/NaGdF4:A  was also improved 20%–30% by successive layer‐by‐layer method. Chen's group reported the synthesis of LiLuF4:Ln3+ core/shell UCNPs through successive layer‐by‐layer method and high absolute upconversion quantum yields were achieved 5.0% and 7.6% for LiLuF4:Er and LiLuF4:Tm core–shell structures, respectively.Heating‐up method has a few shortcomings, such as volatile solvent removal, prolonged heating and the centrifugation and washing of core nanoparticles, as such hot‐injection technique was developed to obtain upconversion core–shell nanostructures.4 NCs via thermal decomposition. Then SNCs as shell precursors were injected into a hot solution of core NCs (defocusing) and dissolved and deposited on the larger core NCs (self‐focusing). Therefore, core–shell nanostructures would be obtained. If the cycle process of defocusing and self‐focusing was repeated, multilayer core–shell nanostructures could form  are dissolved and deposited on the surface of nanocrystal cores with larger size due to their high surface energy. Veggel and co‐workers synthesized core–shell nanostructures through this method.m Figure e–i. The 4:Yb,Er@NaGdF4 multilayer core–shell UCNPs were synthesized for the first time . The size of NaYF4:Yb,Er core upconversion nanocrystals was about 16.3 nm. Diameter of NaYF4:Yb,Er@NaGdF4 core–shell nanocrystals with 4‐layer NaGdF4 shell increased to ≈30 nm. With the shell growth, the uponversion optical property was optimized and improved under the same excitation power. Doping luminescent centers in shells, the core–shell nanostructures could realize up‐down conversion dual‐mode luminescence.The prior three methods are generally used to prepare hydrophobic core–shell upconversion nanostructures. Our group synthesized hydrophilic lanthanide‐doped core–shell UCNPs by microwave assisted polyol processes.2 and TiO2 are the most common inorganic shell materials. The reverse microemulsion method is suitable to coat SiO2 shell on the oleate or OM‐capped UCNPs. Numerous functional groups can be encapsulated in SiO2 shells or be connected to the surface of SiO2 shells for further applications.N‐hydroxysuccinimide ester groups.2) has also attracted massive attention owing to their large surface area and tunable pore size. The nanocomposites combining mSiO2 and UCNPs are very promising for biological imaging, drug delivery PDT and chemical detection. For example, Shi and Bu synthesized azobenzene‐modified UCNP@mSiO2, and load anticancer drug doxorubicin (DOX) into mesopores silica.2 shell on the hydrophobic UCNPs via reverse microemulsion method, and then further coated another dense SiO2 shell via water‐phase regrowth method. Hot water etching, with PVP as protecting agents, is conducted to obtain upconversion core/hollow porous silica shell nanostructures (UCSNs). UCSNs are very conducive in terms of drug delivery. TiO2 shell is also coated on the surface of UCNPs for some particular applications, such as photocatalysis2 layer on UCNPs after modification with a surfactant layer.4:Yb,Tm@TiO2 nanocomposites showed obvious photocatalytic activity under NIR light. Lin coated TiO2 on UCNPs for NIR light triggered PDT and Zhao et al. designed novel UCNP@SiO2@TiO2 nanocomposites for high‐performance dye sensitized solar cells.4:Yb,Tm hybrid nanostructures via a solution method.4:Yb,Er@Ag core/shell nanocomposites and demonstrated that they were a promising upconversion imaging and PTT agent.The shells of nonepitaxial core–shell structures, whose crystal lattices mismatch with those of cores, are usually inorganic materials or noble metals. SiO5In recent years, development of rare earth doped upconversion nanomaterials for various applications ranging from biomedical to electro‐optic field, especially bioapplications has been a research focus, in addition to the study of their light emission mechanism, synthesis routes and optical properties. First of all, the safety of upconversion nanostructures is our common concern. Not only for bioimaging and therapy, some detections also require upconversion nanostructures with biocompatibility and low biotoxicity. Therefore, at the beginning of this chapter, the biosafety of upconversion nanostructures is identified as an issue we need to consider, although it has been reviewed by Capobianco et al. and Li et al.5.1As previously mentioned, UCNPs with large anti‐Stokes shift, weak background interference, and no photobleaching are suitable to detect some target species, such as pH,5.1.14:20%Yb,1.6%Er,0.4%Tm UCNPs for detection and bioimaging of cyanide anion.− anions can change the absorption band of Ir1. When adding CN− anions in the LRET system, the energy transfer was blocked and the upconversion emission was recovered. By comparing the rations of UCL in the absence and presence of CN− anions, low detection limit of 0.18 μM CN− anions could be obtained. They also designed a LRET nanoprobe combining hCy7 (an organic dye) and UCNPs  to detect methylmercury (MeHg+), which would cause language and memory barriers , 540 nm (green), and 650 nm (red), respectively. The hCy7‐UCNPs could emit green and NIR emission in the absence of MeHg+. When meeting MeHg+, the hCy7 was converted into hCy7' that exhibits NIR absorption band (centered at 800 nm) rather than red absorption band (centered at 660 nm). Through detecting the ratiometric upconversion emission at 660 to 800 nm, MeHg+ could be monitored. Vetrone et al. developed a nanothermometer based on poly(N‐isopropylacrylamide) (pNIPAM) modified NaGdF4:Yb,Er UCNPs which combined an organic dye (FluoProbe532A) to detect the temperature at a subcellular level.2+,2+,2,2S,3+,2+,Organic dyes like chromophoric Ru(II) complex (N719) and Ir(III) complex (Ir‐9), which have an absorption band matched well with the upconversion emission, could play as energy acceptor for upconversion turn‐on probe.rs Figure10a.1635.1.24:Yb,Tm@NaYF4 UCNPs as energy donor and MnO2 nanosheet as energy acceptor. MnO2 nanosheet was deposited on the surface of UCNPs by reduction of potassium permanganate solution.2 nanosheet modified UCNPs was quenched due to the formation of LRET between Tm3+ ions and MnO2. But facing GSH, the MnO2 nanosheet was reduced to Mn2+ ions. MnO2‐induced UCL quenching effect was inhibited, and the LRET was terminated, thereby UCL was recovered. The recovered upconversion emission was a function of the concentration of GSH. Furthermore, they monitored the GSH levels in living cells. Liu's group reported a LERT probe for biothiols and few‐atom silver nanoclusters (Ag NCs) as energy acceptor of UCNPs, which was presented in Figure If the absorption bands of inorganic nanoparticles are match with the upconversion emission bands of UCNPs, the distance is suffciently close as well, the LRET process would occur.4 UCNPs for the detection of β‐hCG (an important disease marker)  Figure c.41 Firs5.23+ have two emission bands centered at 540 and 650 nm, separately. So single‐band red upconversion light (650 nm) is a purse for bioimaging.3+‐doped UCNPs with an upconversion emission peak centered at 800 nm under 980 nm, in which both excitation and emission bands are located in the “optical window,” provide a high penetration depth. Prasad et al. demonstrated high‐contrast UCL imaging of deep tissues (UCL was imaged through 3.2 cm pork tissue) based on α‐NaYbF4:Yb,Tm@CaF2 core−shell nanoparticles.3+ as sensitizer was introduced into upconversion nanostructures. Yan's group demonstrated that using shorter wavelength excitation band centered 808 nm rather than 980 nm could greatly minimize the tissue overheating effect.4:Yb,Tm UCNPs and they fed C.elegans with the mixture of B‐growth media and NaYF4:Yb,Tm UCNPs.4 UCNPs for UCL bioimaging in a small black mouse.FigureBioimaging technology has attracted much attention given its ability of visualization. Fluorescence imaging, MRI, and CT are the most common bioimaging techniques. First, we talk about the fluorescent bioimaging based UCNPs. Compared to the traditional fluorescent probes, such as semiconductor quantum dots or organic dyes, UCNPs with some advantages, such as weak background autofluorescence, deep tissue penetration, low photobleaching, and large anti‐Stokes shifts, are good fluorescence agent for in vitro or in vivo imaging. Autofluorescence can be eliminated because bio‐samples do not have upconversion emission under NIR excitation. For bioimaging, it is optimal that excitation and emission wavelengths are in the NIR spectral range (700–1100 nm) and red region (600–700 nm) which is termed “optical window” of the biological tissues. The UCNPs containing Er3+ ions in UCNPs are of importance for some certain imaging techniques. This kind of UCNPs is an excellent multimodal imaging agent. Gd3+ ions with seven unpaired electrons in the ground state show large paramagnetic moment, which is commonly used as T1‐weighted MRI contrast agent. Gd3+‐based host materials, such as NaGdF4,4,4,53+ ions could exist as host matrix or just as a shell coated on core nanoparticles.3+ ions can also be incorporated into the host matrix as dopant. Li and co‐workers reported NaYbF4:Gd,Yb,Er nanophosphors with suitable magnetic properties (r1 = 0.41 s−1 Mm−1) for both MRI and upconversion imaging of Kunming mice.3+ ions by modification using gadopentetic acid (Gd‐DTPA). For example, Li's group obtained a core–shell UCNP‐based nanostructure with NaLuF4:Yb3+, Tm3+ as core SiO2 as shell and Gd complex of Gd‐DTPA as capping surfactant. These nanostructures had excellent UCL, high r1 value of 6.35 s−1 mM−1, and strong X‐ray attenuation, which display relatively low biotoxicity and are for upconversion/MRI/CT tri‐modal bioimaging.3+ or Co3+ ions, also can combine UCNPs as T2‐weighted MRI contrast agent.4:Yb,Tm@FexOy nanostructures which exhibit excellent UCL of NIR to NIR and a saturation magnetization of ≈12 emu g−1.2‐weighted MRI/upconversion bio‐imaging in vivo of the lymphatic system had been done. The design might be of great help to clinical lymph nodal study and diagnosis.In order to achieve the temporary and spatial sensitivity and accuracy of diagnosis, multimodal bioimaging was developed. Multimodal bioimaging based on UCNPs, which combines UCL imaging with other imaging technologies, like MRI and CT, could make up for their own shortcomings and tap their respective advantages. So the multimodal bioimaging has great contribution to further clinical treatment. In addition to UCL, some specific Lnx18F , it has been the most commonly used radionuclide for PET bioimaging. PET bioimaging, which could produce 3D imaging, is used for biodistribution investigation. Li et al. fabricated 18F labeled magnetic‐upconversion nanophosphors for in vivo upconversion/MRI/PET bioimaging.18F, 153Sm with a half‐time of 46.3 h is commonly incorporated into upconversion nanostructures as radionuclide for SPECT imaging. Li's group reported the synthesis of NaLuF4:153Sm,Yb,Tm nanoparticles. Nanoparticles were utilized for in vivo SPECT imaging with high sensitivity and the ex vivo biodistribution of nanoparticles was easily qualified by this method.The lanthanides, especially Lu, with higher atomic numbers than iodine show excellent X‐ray attenuation ability, which makes them an optimal option as CT contrast agent. Obviously, Au,4:Yb,Tm as core and 4 nm of 153Sm3+‐doped NaGdF4 as shell. In this nanostructure, Yb and Tm, Lu, Gd, and Sm153 were responsible for UCL, CT, MRI, and SPECT imaging, respectively. The combination of those four imaging modalities can provide detailed information, which shows its value in tumor angiogenesis imaging.4:Tm@NaYF4 UCNPs. The two active imaging components of PoP and UCNPs could be used in no less than six different imaging techniques, including fluorescence (FL), photoacoustic, Cerenkov luminescence (CL), PET, CT and UCL imaging , with a particle diameter of sub‐50 nm.2 layer via a water‐in‐oil reverse microemulsion technique. Furthermore, in order to improve the efficiency of loading photosensitizers, Zhang's group designed mesoporous silica coated UCNPs and incorporated ZnPc (photosensitizer) into the mesoporous silica shell. Under NIR 980 nm irradiation, the UCNPs converted NIR light to visible light which could further activate ZnPc to produce reactive singlet oxygen for killing cancer cells.−2 of 978 nm light for 45 min. The photosensitizer can also attach to the surface of UCNPs via covalent bonding.4:Yb,Er@CaF2@SiO2‐PS nanostructure for PDT in vitro, in which, photosensitizer (PS) was covalently grafted to mesoporous channels of mesoporous silica.1O2 production efficiency, the 660 nm upconversion emission of NaYF4:Yb,Er UCNPs was enhanced via doping 25% Yb3+ and the efficiency of energy transfer was improved because the distance of energy transfer was shortened via covalent assemblage. Liver tumor inhibitory ratio was ≈80.1% under 980 nm irradiation duration 15 min  has been designed by Shi's group.Compared to surgery, chemotherapy and radiotherapy, PDT is a noninvasive cancer treatment. PDT is involved, in which PDT drugs (photosensitizers) are activated by UV, visible light or NIR to generate cytotoxic reactive oxygen species (ROS) for killing the target cells. The organic molecules, such as methylene blue (MB), zinc(II) phthalocyanine (ZnPc), Chlorine6 (Ce6) and so on, and semiconductor nanomaterials are the most common photosensitizers for PDT. Because UV and visible light with low penetration ability limit the development of PDT, utilizing UV and visible light converted by UCNPs from NIR can overcome the limitation of low penetration ability.in Figure13a,b. 5.3.22, mesoporous SiO2 or polymer coated UCNPs with porous or hollow structure are good drug and siRNA carrier for chemotherapy.5:Yb/Tm UCNPs as drug carrier.2@DOX‐ZnO nanostructure.2. ZnO as a gatekeeper blocked the mesopores of SiO2 to the noneffective leakage of drugs. However, ZnO could be dissolved in acidic solution. So, drugs could be effectively released in the location of the tumor with very low side effects. The multimodality bioimaging including UCL, CT and MRI provided detailed and exact information for drug release. They demonstrated that higher amounts of HeLa cell death caused by UCNP@mSiO2@DOX‐ZnO nanostructures than DOX. Phototrigger‐induced chemotherapy based on UCNPs utilizing the visible or ultraviolet light converted by UCNPs from NIR to control drug release, which means that UCNPs are a switch of drug release. For example, Shi's group reported the synthesis of hollow mesoporous silica coated NaYF4:Yb/Tm@NaGdF4 (UCNP@hmSiO2) nanostructures.2  as drug carrier.Although chemotherapy is invasive, it is still an effective method for disease treatment. The chemotherapy based on UCNPs includes two aspects: imaging‐guided chemotherapy and phototrigger‐induced chemotherapy. Imaging‐guided chemotherapy could indirectly observe and monitor the extent of drug release by using the UCL of UCNPs. SiO2 Figure c,d. The 5.3.33O4 nanoparticles, and Au nanoparticles via layer‐by‐layer self‐assembly to be used for multifunctional bioimaging and PTT.xS were also used as photothermal agents for tumor therapy. Zhang et al. reported the synthesis of GO covalently grafted UCNPs and then loading ZnPc on GO, which could act as a theranostic platform for UCL bioimaging and PTT/PDT of cancer.2O3:Yb/Er−CuxS multifunctional nanostructures. DOX could be loaded into the nanostructures. Simultaneous bioimaging and chemotherapy/PTT could be achieved in this nanostructure, where CuxS acted as the photothermal functional section, DOX acted as the chemotherapeutic drugs and UCL acted as the part of bioimaging.  nanostructures, they utilized Au25(SR)18 clusters with size of 2.5 nm to produce PDT and PTT effect by receiving energy from the UCNPs and the pH/temperature‐responsive P(NIPAm‐MAA) could control DOX release. An in vivo anticancer therapy has been conducted, which demonstrated that this design could markedly improve the therapeutic efficacy.In many cases, these three treatments are used together.5.42 is a semiconductor, which is frequently used for solar energy harvesting. However, TiO2 with a high band gap (3.2 eV) can only absorb UV light and most of the solar energy is wasted. Therefore, incorporating upconversion materials and TiO2 into the system of solar energy harvesting, where TiO2 absorbs the visible or ultraviolet light converted by upconversion material from NIR, can increase the utilization of solar energy.t‐BOC‐coated α‐NaYF4:Yb/Er and α‐NaYF4:Yb/Tm UCNPs.4 upconversion microrods, which could act as multicolor barcoding for anticounterfeiting.4:Yb/Tm UCNPs to develop a new temporal‐domain approach to multiplexing.4:Yb,Er,Gd UCNPs and demonstrated that the UCNPs could be in 3D displays.In addition to the above applications demonstrated, upconversion nanostructures could also be used for solar energy harvesting,6In this review, we reviewed recent advances in upconversion nanostructures in terms of mechanism, design and synthesis, and some applications. We also provided a number of typical examples to demonstrate the various aspects of upconversion nanostructures in detail. Though, the development of upconversion nanostructures grows substantially in recent years, critical challenges remain, which impose great barriers to further optimize the upconversion nanostructures for commercialization. First, controllable preparation of upconversion nanostructures is highly desired, which includes both morphology and composition, and surface properties and the ability for chemical modifications. As one kind of optical materials, the main issue of upconversion nanostructures is that the fluorescence efficiency of the upconversion is far less sufficient, which largely limits their applications. One urgent task is to generate upconversion nanostructures with high fluorescence quantum yield. Obviously, upconversion is a nonlinear optical phenomenon, moreover, quantum yield is not applicable for characterizing their fluorescence efficiency. Therefore, it is required to establish a new and sound pathway to evaluate upconversion fluorescence efficiency. In addition to fluorescence efficiency, the color of upconversion emission, such as single‐band and full‐color emission, should be a research focus. On the other hand, the dispersibility, chemical stability, biocompatibility, and long‐term toxicity are still issues that should be well addressed in the development process. Last but not the least, the upconversion nanostructures should be combined with other functional materials to create new and promising structures and properties.


Pseudomonas aeruginosa PAO1, a facultative denitrifying bacterium and an opportunity pathogen, was used to study antibacterial assays under both anaerobic and aerobic conditions. The results indicate that the respiration status of P. aeruginosa affect the ecotoxicity of AgNPs. P. aeruginosa cultured under aerobic condition were more susceptible to AgNPs than that under anaerobic condition. Aerobic condition greatly enhanced bacteriostatic effects of AgNPs but not their bactericidal effects, as the ratio of viable but nonculturable (VBNC) bacteria remained above 90% when 5 mg L−1 AgNPs applied. Our findings offer further understanding for the degree of toxicity of nanoparticles on microbial ecosystems and underscore the importance of exposure condition (e.g. oxygen) in the mode of action of AgNPs.The intensive applications of silver nanoparticles (AgNPs) inevitably cause continuous release of such materials into environments, as a consequence posing potential risks to microbial communities in engineered or natural ecosystems. However, the magnitude of antibacterial capacity of nanoparticles is still inconclusive, owing to influential factors such as the size of nanoparticle, microbial species, or environmental conditions. To reveal whether the presence of air would alter AgNPs ecotoxicity,  AgNPs are the most popular nanoparticles in markets, which accounts for about 50% of commercial nanoproducts4. It is estimated that approximate 500 tons of AgNPs were produced each year worldwide6. As being incorporated in fabric and personal care products, the processes of fabrication and consumption thereby can release AgNPs from consumer products to sewage, eventually entering environment. Thus, it represents a potential risk to a great variety of organisms in natural or engineered ecosystems, including wastewater treatment plants (WWTPs).Silver nanoparticles (AgNPs) have been widely used in consumer products such as textile, cosmetics, and medical products, due to their excellent antibacterial, optical, and electronic propertieset al.7 reported that the respiration of autotrophic nitrifiers in a continuously stirred tank reactor was inhibited by 86% at 1 mg L−1 of AgNPs. Tan et al.8 documented that AgNPs at 300 µg L−1 decreased nitrifying efficiency from 98% to 15%. In addition, Li et al.9 showed that a commercial AgNPs at 10 mg L−1 was the miminum inhibitory concentration (MIC) to Escherichia coli, whereas a synthesized AgNPs exhibited 100% killing of E. coli when added at 4.5 mg L−110. According to these previous studies11, the concentration of AgNPs that will cause toxic impacts on microbes, ranged from µg L−1 to mg L−1. Such an inconclusive antibacterial capacity of nanoparticles may be associated with different exposure conditions or microorganism types, which may alter the antibacterial activity of nanoparticles. Some studies15 suggested that the susceptibility of microbes, microbial adaptability, the synthesis and coating of NPs, and dissolution of metal cations in aqueous solution environment would affect the ecotoxicity of AgNPs. For instance, Lu et al.15 investigated the antibacterial effects of AgNPs by exposure of five oral pathogenic bacteria to AgNPs, and corresponding MICs varied from 25 to 50 mg L−1. Arnaout and Gunsch16 tested impacts of AgNP coatings  on Nitrosomonas europaea, and found that citrate AgNPs had a stronger inhibition on nitrification and caused more serious membrane disruption, compared to AgNPs coated with gum Arabic or polyvinylpyrrolidone.AgNPs have shown great antibacterial activities against various microbes. Choi + can be chemisorbed, or liberated from AgNPs into liquid especially in the presence of oxygen17. Thus, the aerobic and anaerobic conditions may interfere with the effects of AgNPs on microbial activities through changing the dissolved Ag+ in their living environment18. There seems a general agreement in previous studies20 that the dissolved Ag+ is the factor leading to the toxicity of AgNPs. However, both anaerobic and aerobic conditions exist in natural or engineered ecosystems, thus microbial communities would be easily exposed to these two conditions. As most bacteria in WWTPs are facultative bacteria, it is of great importance to investigate the effects of anaerobic and aerobic conditions on the difference of antibacterial effects of AgNPs to facultative microorganisms, considering previous studies reported differential degree of toxicity.Although the ecotoxicity of AgNPs has attracted more and more attention, it is so far still not clear if different respiratory conditions (aerobic vs anaerobic) can affect the toxicity of AgNPs. Zero-valent AgNP is insoluble in water, but AgPseudomonas aeruginosa PAO1 (P. aeruginosa), a ubiquitous denitrifier in WWTPs contributing to nitrogen removal and also an opportunistic pathogen often found in hospital environment21, was used as a model bacterium to evaluate how exposure condition affects the action of AgNPs. Their dose-response patterns are compared under anaerobic and aerobic conditions based on antibacterial assays using physiological and metabolic approaches.The objective of this study is to examine the response of environmental bacteria to AgNPs, and to compare the toxicity of AgNPs under anaerobic versus aerobic conditions. Pure culture investigation can give us insights into the potential mechanisms of AgNPs antibacterial action toward microbes. Therefore, 600  profiles were monitored to refelect the status of cell growth . Furthermore, the growth of P. aeruginosa was totally inhibited in the presence of 5 mg L−1 AgNPs, irrespective of the presence or absence of oxygen.To investigate antibacterial property of AgNPs under anaerobic versus aerobic conditions, ODwth Fig. . In the P. aeruginosa, which were consistent with OD profiles  and cell growth rate (4.1 × 106 cells mL−1 h−1) were observed in aerobic controls compared to that  in anaerobic controls (p > 0.05). P. aeruginosa cultures amended with AgNPs at a concentration of 1 mg L−1 also obtained a faster growth rate under aerobic respiration, albeit this dosage level ocuured lag phase extension. However, this trend reversed with the addition of AgNPs of 2.5 mg L−1, which caused a stronger inhibition on cell proliferation under aerobic condition. It is noted that with the increasing addition of AgNPs from 1 to 2.5 mg L−1, bacteria cultured anearobically exhibited slight increase in cell growth rate (from 9.1 × 105 to 11.2 × 105 cells mL−1 h−1) whereas those cells cultivated aerobically declined significantly (from 2.7 × 106 to 2.6 × 105 cells mL−1 h−1). It implies that 1 and 2.5 mg L−1 of AgNPs have similar inhibitory effects on cell growth under anaerobic condition. However, aerobic condition exerts an increased stress on cell growth with the AgNPs concentrations. Consistent with OD profiles, flow cytometry resutls showed that AgNPs at 5 mg L−1 completely hindered cell growth under both conditions as the total cell number remained at the same level during 24 h exposure.The results based on flow cytometry Fig.  further les Fig. . For con−1 AgNPs. Under exposure of 2.5 mg L−1 AgNPs, the live cell ratio was still high (99%) in anaerobically cultivated cells, wheras that of the aerobically cultivated cells encountered a decline (93%). With the AgNPs concentration being increased to 5 mg L−1, the live cell ratio descreased to 95% under anaerobic condition, while that kept at 93% under aerobic condition. The dead cell ratios of treated cultures were shown in Fig. −1; while AgNPs at 5 mg L−1 caused shut down of cell multiplication of PAO1 but did not affect cell viability to the same extent, indicating cells were in viable but nonculturable (VBNC) status.In order to further understand the physiological status of cells in various cultures, LIVE/DEAD staining was performed to observe cell viability Fig. . High li23. The FSC vs. SSC density plots of cells in controls and cultures amended with AgNPs (2.5 mg L−1), under anaerobic or aerobic conditions, were demonstrated in Fig. During the FACS (fluorescent-activated cell sorting) detection process, cells were examined by FSC (forward scatter) and SSC (side scatter) detectors and represented in SSC distribution Fig.  and FSC −1 after 33 h cultivation, compared to 25.4 mM L−1 in anaerobic control (p > 0.05). Although the dosage of 1 mg L−1 AgNPs extended the lag phase of P. aeruginosa growth under aerobic condition, its glycerol consumption (27.0 mM L−1) exceeded anaerobic control (25.4 mM L−1) after 33 h cultivation, consitent with OD600 values . AgNPs at 5 mg L−1 totally hindered cell growth under both conditions, with very negligible carbon source consumption.To assess how the AgNPs influence bacterial metabolism, carbon source consumption was monitored during the exposure period. Consistent with the cell growth profiles, the glycerol consumption Fig.  decrease−1) were noticeable. The addition of AgNPs resulted in membrane damage of P. aeruginosa cells  treated cultures.To understand alterations in cell morphology or structure after AgNPs treatment under anaerobic and aerobic conditions, TEM images Fig.  of contrlls Fig. . Controllls Fig.  have a s+ to biological effects is challenging due to their common co-occurrence24. To reveal potential involvement of silver ions liberated from AgNPs on cell growth inhibition, the levels of Ag+ released in the media were determined . Almost all exposure conditions obtained similar amount of Ag+ release (i.e. 0.110 ~ 0.124 mg L−1) after 24 h exposure  also have strong ecotoxicity under anaerobic condition. Lu et al.15 also reported that AgNPs exhibited some antibacterial activity against five anaerobic oral pathogenic bacteria by dosing 25 ~ 50 mg L−1 as the minimum inhibition concentration (MIC). In contrast, some studies suggested that AgNPs generally would not affect the performance of anaerobic biological treatment systems25, since an anaerobic bioreactor may prevent silver ion release18 or transform AgNPs into silver sulfide26, which may provide detoxification mechanisms for AgNPs.It is also observed that AgNPs inhibited bacterial activities under anaerobic condition, indicating the general toxicity of AgNPs. The repression shown in bacterial growth Figs  and 2 an−1, cell growth was eventually not displayed, implying that the increasing AgNPs dosage did not correspond to an increase in cell death ratio but accelerated the transition to VBNC status. Similar results were found when performing high concentrations (2 mg mL−1) of TiO2-NPs to Pseudomonas putida, that nearly all the cell population were viable but only one third of them were cultivable27. It has been previously reported that the VBNC state plays an critical role in the survival of bacteria, and in their ability to affect environment and human health29. In VBNC cells, the transport and biosynthesis system, and the ability to utilize substrates are maintained. It is usually accompanied by a reduction in metabolic activity levels to minimize cellular energetic requirements30, which is an survival strategy of many bacteria in response to adverse environmental conditions. Thus, the resuscitation of VBNC cells would allow them regain cultuability and, as P. aeruginosa a pathogen, renewed ability to induce infection28. Further studies on these VBNC cells resulted from the stress of AgNPs would be interesting.Although 93% of the total cells were still viable in suspensions amended with AgNPs of 5 mg L+ leaching levels from AgNPs was performed to determine whether Ag+ associated with the differential displayed toxicity of NPs under anaerobic versus aerobic conditions. Table + was not the key determinant that caused the different ecotoxicity between two culturing conditions. The anaerobic condition was supposed to eliminate confounding effects associated with oxidative release of silver ions13. However, except oxidative dissolution of AgNPs caused silver ion, the chemisorbed silver ions from the surface of AgNPs is another Ag+ release mechanism17. The synthesis approaches may also affect the silver ions distribution in the media. In the present study, released Ag+ was detectable under anaerobic conditions at the beginning of exposure (2 h) even at lower dosage level (1 mg L−1), and the amount of leaching Ag+ reached to the same level with that under aerobic condition after 24 h. Specifically, AgNPs at 2.5 mg L−1 released comparable amount of Ag+ under aerobic and anaerobic condition at 2 or 24 h exposure. However, this dosage induced a stronger inhibition on aerobic cell growth in terms of OD and live cell numbers, compared with anaerobic condition. In addition, under anaerobic condition, similar levels (0.115 ~ 0.118 mg L−1) of Ag+ were detected when exposed to AgNPs of 2.5 and 5 mg L−1 for 24 h, while 5 mg L−1 of AgNPs appeared to be more toxic to P. aeruginosa. These results indicate that anaerobic condition could not eliminate Ag+ release from AgNPs as expected, and the released Ag+ was not the dominant bacteriostatic mechanism of AgNPs, which is different with the previous report suggesting that Ag+ released from AgNPs was mainly responsible for the toxicity of AgNPs18.The detection of Ag31. Oxygen condition may increase the intracellular ROS production in cells4. In this study, ROS detection assay was conducted, estimated by 2′,7′-dichlorofluorescindiacetate , to check variations of cellular ROS. However, treated cells had no distinct difference in ROS level compared to the controls (data not shown). So it could rule out ROS induction as a possible mechanism for acute toxicity of AgNPs to P. aeruginosa. Yang et al.20 also did not detect ROS induction when exposed Pseudomonas stutzeri, Azotobacter vinelandii, and Nitrosomonas europaea to AgNPs with 0.5 mg L−1. Another proposed explanation is the abrasive surface texture of AgNPs14, which has more uneven and rough surface texture when exposed to air24. This surface roughness may relate to the mechanical damage of cell membrane as shown in TEM images  on cellular constitutes and cell membrane−1 exhibited an enhanced ecotoxicity to P. aeruginosa under aerobic condition, suffering inhibition on cell proliferation and lower cell viability in terms of flow cytometry results. Correspondingly, carbon source utilization encountered more suppression under aerobic condition. In addition, AgNPs also exert antibacterial influences when bacteria grow anaerobically. Physiological changes in P. aeruginosa were observed with the transition of cells to VBNC status when AgNPs posed inhibition on bacteria. Results obtained from ICP-MS detection revealed that silver ion has a negligible contribution to the detrimental performance of AgNPs in this study. The findings offer improved understanding on the magnitude of AgNPs toxicity on microbial ecosystems and underscore the importance of aqueous solution environment (e.g. oxygen) in the mode of AgNPs antibacterial activity.In summary, this study documented that aerobic condition enhances bacteriostatic property of silver nanoparticles, compared to anaerobic condition. AgNPs at 2.5 mg L−1, purchased dispersion was diluted in Milli-Q water and homogenized by ultrasonication (80 W) for 1 h in water bath (20 °C). These particles have a mean size of 40 nm in the stock dispersion, determined by dynamic light scattering with a Zetasizer Nano ZS .Silver nanoparticles dispersion  was obtained from Fraunhofer, Germany. The AgNPs were synthesized by an aqueous reduction method. To make AgNPs stock dispersion of 1 mg mLP. aeruginosa strain (DSM No: 22644), as the model microorganism for AgNPs experiments, was obtained from the Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Germany. Following the supplier’s instructions, the stain was activated by growing on tryptic soy agar (TSA) plates at 30 °C for 26 h. A single colony on TSA plates was inoculated to tryptic soy broth (TSB) and grown in a shaking incubator  overnight. Afterwards, bacterial suspension (5 mL) in TSB was transferred into serum bottles fed with 150 mL of sterile anaerobic glycerol modified M9 (GLYM9) media32 in an anaerobic chamber and incubated overnight. The optical density at 600 nm (OD600) of this culture was then adjusted to 0.5 and multiple 10 mL aliquots were transferred respectively to each serum bottle and cell culture flask  containing 150 mL sterile GLYM9 medium. All cultures were incubated at 30 °C.−1 glycerol, 30 mM NaNO3, 0.2 g L−1 yeast extract, 0.2 M phosphate buffer, 9 mM NaCl, 38 mM NH4Cl, 2 mM MgSO4·3H2O, and 100 µM CaCl2·2H2O32. To prepare aerobic media, GLYM9 were autoclaved and then using the biosafety cabinet to transfer media into sterile cell culture flasks with vent cap (Corning), which has 0.2 µm pore hydrophobic membrane allowing air exchange meanwhile minimizing contamination. For anaerobic conditions, GLYM9 media were decanted into serum bottles and followed by sparging with nitrogen gas for 30 min before autoclave sterilization.The ingredients of GLYM9 were 10 g L7 cells mL−1. To observe the interactions between AgNPs and bacteria, various volumes of AgNPs stock dispersion were injected in 150 mL GLYM9 media, immediately after the inoculation of bacteria, to obtain different starting concentrations of 1, 2.5 and 5 mg L−1. These concentrations of AgNPs were chosen on the basis of preliminary studies (data not shown), expressing the spectrum of cell growth responses to AgNPs. Control cultures had no amendments. P. aeruginosa cultures were further incubated for 48 h under either anaerobic or aerobic conditions. All experiments were performed in triplicate. During the exposure period, samples were collected from triplicate cultures at nine different time points  for determination of cell growth and viability, and supernatants were used for determination of glycerol and silver ions levels, as described below.Sterile culture media in serum bottles (anaerobic) and flasks with vent cap (aerobic) were inoculated, in anaerobic chamber or biosafety cabinet respectively, with bacterial suspension from overnight pre-cultures to achieve an initial cellular concentration of about 10600, as an indicator of cell density, was measured during the exposure period by using a Cary 50 bio UV visible spectrophotometer . The final concentration of AgNPs in the media resulted in an acceptable scattering error contribution (<10%) for the OD600 absorbance (<0.01). Cell density and viability of P. aeruginosa, non-amended and amended with AgNPs, were measured using LIVE/DEAD staining . In brief, triplicate cultured cells were stained with 1.5 µL each of SYTO 9 (S9) and propidium iodide (PI) per 1,000 µL sample, and incubated in the dark for 15 min at room temperature before the measurement by a FACSAriaTM II flow cytometer . The determinations of cell concentrations in suspensions were performed by adding fluorescent microspheres (100 beads per 1 µL). Thus, the cell counts of D. vulgaris can be calculated by the ratio of bacterial events to microsphere events. To determine Live/Dead ratio of cells, cells located in specific gate were expressed as a proportion of total cells, as described in the previous study21. FACSAriaTM II flow cytometer  contains 5 lasers, two of which (Blue 488 nm and Yellow/Green 561 nm) were used in this study, (forward scatter) FSC detector and (side scatter) SSC detector. As each cell in samples untercepts the laser beam, laser light is scattered in all derections. The FSC parameter was used as an indicator of cell size, whereas SSC parameter was used to indicate cell complexity27.ODP. aeruginosa. The suspensions of P. aeruginosa cultures were immediately filtered through 0.22 µm filters . Glycerol concentrations were detected by a high-performance liquid chromatography (HPLC), which is a Shimadzu LC equipped with an Agilent Hi-Plex H column (300 × 7.8 mm) specialized for organic acid analysis.During the cultivation period, the concentration of glycerol was monitored to reflect the metabolism of P. aeruginosa cells amened with AgNPs under aerobic versus anaerobic conditions, suspension samples were collected at 6 h cultivation and directly centrifuged at 5,000 rpm for 5 min. Samples for TEM imaging were performed by standard procedures, and details are similar with the previous study34. Briefly, after discharging supernatants, pellets were resuspended by agar and deposited on a copper grid, immediately frozen via a high-pressure freezer and then freeze-dried. After embedded in epoxy resin, ultrathin sections (50 ~ 100 nm) of samples were then cut and dropped off on TEM copper grid and imaging was performed by TEM using a JEOL JEM-1011  operated at 80 kV.To compare the different morphology of 26. Filtrates were brought to containers with 1 mL of concentrated nitric acid to acidify and digest metal cations to measure total dissolved silver ions.Inductively coupled plasma mass spectrometry (ICP-MS) (Agilent 7700) was performed to quantify silver ions leaching from AgNPs under both aerobic and anaerobic conditions. Aliquots of media amended with AgNPs under different culture conditions were centrifuged . Supernatants were then filtered through centrifugal filter  to capture nanoparticlesResults are presented as mean (± standard deviation) from triplicate experiments. Statistical significance was determined by one-way analysis of variance (ANOVA) for the total cell counts and viability.The datasets generated during and/or analysed during the current study are available from the corresponding author on reasonable request.


MMI) and elucidate their underlying regulatory mechanisms.We aimed to explore potential cytokines involved in the malignant middle cerebral artery infarction . The differentially expressed cytokines were then identified in patients with MMI using two‐tailed Student's t‐test and Fisher's Exact Test compared with patients with NACI. Gene Ontology and pathway enrichment analyses were performed using DAVID. Protein–protein interaction (PPI) network was constructed based on STRING database.We first developed a cytokine profile by QuantibodyNCAM1 , IGFBP‐6 (insulin‐like growth factor binding protein 6), LYVE1 , and LCN2 , were up‐regulated, while another six cytokines, such as TGFB1 , EGF , PDGFA , MMP‐10 , IL‐27 (interleukin 27), and CCL2 (chemokine (C‐C motif) receptor 2), were down‐regulated. Moreover, cytokine–cytokine receptor interaction pathway was significantly enriched.A total of 10 differentially expressed cytokines were identified from 320 unique inflammatory cytokines in serums. Among them, four cytokines, like NCAM1, LCN2, IGFBP‐6, LYVE1, MMP‐10, IL‐27, PDGFA, EGF, CCL2, and TGFB1 may participate in the development of MMI. Moreover, cytokine–cytokine receptor interaction pathway may be an important mechanism involved in this disease. These differentially expressed cytokines may serve as diagnostic biomarkers or drug targets for MMI.Our findings indicate that 10 differentially expressed cytokines, such as  Then the differentially expressed cytokines in serum of patients with MMI were identified. Besides, functional enrichment analyses and protein–protein interaction (PPI) network analysis were performed. The objective of our study was to explore potential cytokines involved in the MMI and to elucidate their possible regulatory mechanisms in MMI development.22.1A total of eight patients with MMI and eight patients with NACI were recruited. MMI was diagnosed according to the following criteria: signs on CT showed an infarct of at least 50% of the middle cerebral artery territory or infarct volume >145 cm³ on diffusion‐weighted images (DWIs); secondary neurological deterioration including consciousness decline was defined by at least 1 point of consciousness item described in the National Institutes of Health Stroke Scale (NIHSS), which was an essential parameter to assess the severity of acute ischemic stroke; patients were hospitalized within 24 hr of disease onset; patients had no history of other brain disorders, such as hemorrhagic cerebral vessel disease, idiopathic epilepsy, intracranial infection, or craniocerebral trauma; and patients had no other infections such as urinary tract infections or pneumonia. Meanwhile, NACI was defined as non‐acute cerebral infarction (7 to 14 days after onset) and had no any clinical sequelae after 6 months. The infarct volume of NACI patients was confirmed according to T2‐weighted magnetic resonance imaging. In addition, the clinical baseline data of the above two different types of diseased population was shown as follows: the median age was 68 ± 3.36 (range: 57–80) vs. 70.75 ± 2.51 (range: 61–79) years, the ratio of male/female was all 1:1. The risk factors of patients are shown in Table g for 15 min. Finally, the serum fraction was separated, aliquotted, and stored at −80°C for further use. Our study was approved by Ethics Committee of General Hospital of Shenyang Military Area Command and written informed consent was obtained from each patient or their responsible families.The blood collection of the included MMI patients was performed at 24 hr of stroke onset. The blood samples of NACI patients were collected at the next morning after admission. The serum collection from these patients was performed as perviously described. In brief, 10 milliliters of peripheral blood sample was collected from median cubital vein of each individual and then centrifuged at 1000 2.2® Human Cytokine Antibody Array 7000, RayBiotech, Inc., Norcross, GA, USA) was used to detect multiple cytokines simultaneously in serum of patients with MMI (n = 8) and patients with NACI (n = 8). This array system utilized the multiplexed sandwich ELISA‐based technology to quantitatively measure the concentration of 320 human cytokines. Briefly, an equal amount of sample diluent was added into each well and incubated for 30 min to block slides. Then multiple cytokine‐specific capture antibodies together with the positive controls were arrayed onto the glass support in quadruplicate. After incubation for 2 h with gentle shaking, the diluted serum samples were incubated with the second biotinlabeled specific detection antibody to recognize a different isotope of the target cytokine. The streptavidin‐labeled Cy3 equivalent dye was then added to incubate in dark room for 1 h, and the signals were subsequently visualized using a laser scanner. Data extraction was done using microarray analysis software GenePix laser scanner , and Quantibody® Q‐Analyzer software  was used for quantitative data analysis. By comparing signals from positive controls, the cytokine concentration in each serum was finally determined based on the standard curves generated by 8‐point cytokine concentrations.An antibody‐based cytokine array system (Quantibody2.3t‐test or Fisher's Exact Test (χ2) test with SPSS version19.0  . The differentially expressed cytokines in serum of patients with MMI were then identified compared with patients with NACI. A value of p < .05 was considered statistically significant. Moreover, the fold change in the individual cytokine expression in serum of patients with MMI compared to that in serum of patients with NACI was calculated to distinguish up‐ and down‐regulation of cytokines. Fold change >1 represented that this cytokine was up‐regulated in serum of patients with MMI compared with patients with NACI, while fold change <1 represented down‐regulation.Analysis of continuous variables and categorical variables between these cytokines were, respectively, carried out using the two‐tailed Student's 2.4http://www.geneontology.org)      , IGFBP‐6 (insulin‐like growth factor binding protein 6), LCN2 , and LYVE1  were up‐regulated, while another six cytokines, like TGFB1 , MMP‐10 , IL‐27 (interleukin 27), EGF , PDGFA , and CCL2 (chemokine (C‐C motif) receptor 2, also known as MCP‐1), were down‐regulated.3.3We performed GO enrichment analysis for functional annotation of these differentially expressed cytokines. As shown in Table In addition, the significantly enriched KEGG, Reactome and PID pathways of these differentially expressed cytokines were shown in Table Besides, the detailed pathway information of cytokine–cytokine receptor interaction is shown in Figure 3.4Based on the information of STRING database, the PPI network constructed by the above 10 differentially expressed cytokines was obtained Figure a. The re4Microarrays analysis has been shown to measure the concentrations of many proteins simultaneously, which dramatically accelerates the pace of discovery of drug targets and biomarkers  ischemic stroke. A comparison of MMI patients with patients suffering from an acute  ischemic stroke would clarify much better the significance of the measured cytokines regarding the diagnostic relevance of MMI. Further studies with larger sample sizes and experiments are warranted to validate our microarray results.Utilizing microarray analysis to detect key cytokines associated with MMI quickly and simultaneously is an obvious advantage of our study. This method will provide a powerful tool for exploring drug targets and diagnostic biomarkers. However, small sample size was the limitation of our study. Moreover, due to lack of clinical samples of MMI and NACI, these key cytokines that we obtained were not confirmed by different molecular assays, such as ELISA, western blot analysis, or other In conclusion, we quantified the levels of 320 cytokines in peripheral blood of patients with MMI and patients with NACI, and 10 differentially expressed cytokines, such as NCAM1, LCN2, IGFBP‐6, LYVE1, MMP‐10, IL‐27, PDGFA, EGF, CCL2 and TGFB1 might contribute to the development of MMI. Moreover, cytokine–cytokine receptor interaction pathway may be an important mechanism involved in this disease. These differentially expressed cytokines may serve as diagnostic biomarkers or drug targets for this disease.None.All authors had full access to all the data in the study and take responsibility for the integrity of the data and the accuracy of the data analysis. Study concept and design: Yaling Han, Huisheng Chen, Zhonghe Zhou. Acquisition of data: Zhonghe Zhou, Jinghua Zhang, Xiaoqiu Li. Analysis and interpretation of data: Cheng Xia, Yaling Han. Drafting of the manuscript: Huisheng Chen. Critical revision of the manuscript for important intellectual content: Yaling Han.We quantified the levels of 320 cytokines in peripheral blood of patients with MMI and patients with NACI, and 10 differentially expressed cytokines, such as NCAM1, LCN2, IGFBP‐6, LYVE1, MMP‐10, IL‐27, PDGFA, EGF, CCL2, and TGFB1 might contribute to the development of MMI. Moreover, cytokine–cytokine receptor interaction pathway may be an important mechanism involved in this disease.


Scientific Reports6: Article number: 3774510.1038/srep37745; published online: 12012016; updated: 03092017The Acknowledgements section in this Article is incomplete.“Genotyping Data used in this work were produced through the technical facilities of ISEM (Institut des Sciences de l’Evolution-Montpellier) and Labex Centre Méditerranéen Environnement Biodiversité. We thank L. Prezeau, E. Valjent and J. Shykoff for comments. MR was funded by grants from CNRS, MW by grants from IUF (Institut Universitaire de France) and Axa Research Foundation”.should read“Genotyping Data used in this work were produced through the technical facilities of ISEM (Institut des Sciences de l’Evolution-Montpellier) and Labex Centre Méditerranéen Environnement Biodiversité. We thank L. Prezeau, E. Valjent and J. Shykoff for comments. MR was funded by grants from CNRS, MW by grants from IUF (Institut Universitaire de France) and Axa Research Foundation. We are very grateful to Franck Lavigne, both for his personal involvement and for coordinating the SEDIMER project (sponsored by the AXA fund). We would also like to thank the students of UGM for their assistance for collecting the data and facilitating our field work”.


Diabetes is one of the most common metabolic disorders, with a high prevalence of patients with poor metabolic control. Worldwide, evidence highlights the importance of developing and implementing educational interventions that can reduce this burden. The main objective of this study was to analyse the impact of a lifestyle centred intervention on glycaemic control of poorly controlled type 2 diabetic patients, followed in a Community Care Centre.A type 2 experimental design was conducted over 6 months, including 122 adults with HbA1c ≥ 7.5%, randomly allocated into Experimental group (EG) or Control Group (CG). EG patients attended a specific Educational Program while CG patients frequented usual care. Personal and health characterization variables, clinical metrics and self-care activities were measured before and after the implementation of the intervention. Analysis was done by comparing gains between groups (CG vs EG) through differential calculations (post minus pre-test results) and Longitudinal analysis.p < .001) and 4% more (effect-size r2 = .04) in BMI (p < .05). When controlling for socioeconomic characteristics and comorbidities that showed to be associated to each parameter in pre-test, from pre to post-test only EG participants significantly decreased HbA1c ; BMI values ; systolic Blood pressure  and diastolic Blood pressure .Statistical differences were obtained between groups for HbA1c and BMI: EG had a decrease in 11% more (effect-size r2 = .11) than CG for HbA1c . Diabetes, one of the most common metabolic disorders, is a public health concern rising with a widespread significance all over the world, with a clear tendency to increase in the next decades –4.1c) in Nacional Health Service (NHS), 21.8% have values over 8% . By analysing the outcomes of this procedure, new contributions regarding the effects of a structured educational intervention program on metabolic control among Portuguese poorly controlled people with type 2 diabetes are highlighted. More specifically, we intend to ascertain whether a multicomponent educational program focused on lifestyle management strategies: (a) is significantly associated with metabolic control; (b) is effective on improving metabolic control; and (c) whether metabolic control is maintained after controlling for socioeconomic and health individual variables (comorbidities).After a piloting procedure , 17, the1c ≥ 7.5%/58.5 mmol/mol), followed in a Community Health Centre of the Portuguese NHS, using age and HbA1c as relevant control selection variables.The sample is composed of two groups of adult/middle age patients with type 2 poorly controlled diabetes (HbAn = 151) were invited during a routine consultation to participate in our study. From those, 136 agreed to do so, and were randomly allocated by the researcher into EG or CG in series of 2 patients for each group . The researcher was the only one in possession of the process number of the patient, without no further information  and q   with HbA1c between 7.5% (58.5 mmol/mol) and 14% (129.5 mmol/mol)  and 58 in CG, between 35 and 67 year-old ; with HbA1c between 7.5% (58.5 mmol/mol) and 12.4% (112 mmol/mol) .Of the 136 participants, 122 concluded all the phases of the procedure : 64 in EG, between 35 and 67 year-old , apart from personal and health characterization variables. Self-care activities were assessed using the Summary of Diabetes Self-care Activities Scale .As for BP as dependent variables, when we control covariates associated with diastolic and systolic blood pressure, the interactions between within-subjects variables and the between-subjects factors were significant, due to a decreased in both groups from pre to post-test,  as for CG we obtained: BPs within-subjects variables: Wilks’ ʎ = .954; F = 2.67; p = .108; ηp2 = .046; observed power = .362 and BPd within-subjects variables: Wilks’ ʎ = .918; F = 4.72; p = .34; ηp2 = .082; observed power = 569 . The same applies to BMI variation, another cornerstone for diabetes health related outcomes, although this variation has a minor estimated decrease and its clinical value may be of lower significance. Blood pressure was the other clinical metric analysed in this study, due to the well-known relation between hypertension and diabetes, a condition that often affects people with type 2 diabetes. Results show both systolic and diastolic values have decreased from pre to post test, although without statistical significance.Impact of the educational program by itself is supported in the Longitudinal analysis, when variables statistically associated with clinical metrics in pre-test measures were controlled. Notwithstanding the statistical removal of the effect of the covariates, a significant interaction between within-subjects variables and the between-subjects factor remains, leading to the conclusion that only in experimental group participants a significantly decreased in clinical metrics can be verified, as identified in other studies .1c values with an effect size of 11%; of BMI, although effect size is lower (3%) and of BP . CG participants didn’t have a significant decrease in HbA1c values (p = .550), in BPs (p = .108) or in BPd (p = .34) and BMI increased from pre to post-test, although it didn’t have statistical significance (p = .782). These outcomes also are in line with the ones from other studies [With distinct effect sizes, we confirmed experimental group participants reduced significantly clinical parameters from pre to post-test. EG participants registered a statistical significant decrease of HbA studies , 39–41.Furthermore, our results sustain the impact of a structured multicomponent educational intervention on improving clinical metrics, beyond standard educational approach alone , 43–45, [ [It is known that often health expected outcomes fail to meet guidelines, patients’ expectations and needs, due to multiple known barriers to health professionals’ intervention and patients’ outcomes achievement[ , 36–38.Education and lifestyle modification are identified as critical components for metabolic and clinical control in diabetic patients. Evidence-based reports have emphasized the importance of education for type 2 diabetic patients and educational programmes have confirmed their role in glycaemic control and decreasing diabetes associated complications ., were identified and taken into account in the definition of the Complex Intervention.The accuracy and innovative contribution of the present study is intended to be the assessment of the effectiveness of a specific educational program, designed upon an exploratory trial, where detailed educational needs of a group of Portuguese people with type 2 diabetes1c with statistical evidence between groups (CG vs EG), result reinforced when correlated variables were controlled.The multicomponent educational program, structured on the principle of Therapeutic Education, has proven its effectiveness, especially with regard to metabolic control, by lowering HbA1c levels between groups (EG and CG). There was even an aleatory patient distribution into groups, which leads us to suggest more research with Portuguese patients, analysing the impact of these programs in health gains, especially if randomization can be achieved.Nevertheless, these results should be interpreted with some consideration due to the limitations of the study, mainly related to the baseline difference in HbA