Functionalized Upconversion Nanoparticles Research Articles

Alginate-functionalized and 4T1 cell membrane-coated multi-tasking nanoparticulate system for near-infrared-triggered photodynamic therapy on breast cancer: In vitro cellular and in vivo mice models

Recently, combination treatment of chemo-dynamic therapy (CDT) and photodynamic therapies (PDT) is well-known and prominent therapeutic strategy for breast tumor. Importantly, tailored nanomedicines with unique personalized properties including nanocarrier suitability and effective targeting ability that requires the exhaustive understanding of tumor microenvironment. Recently, researchers have designed a smart upconversion nanoparticles (UCNPs) that have unique characteristics to achieve targeting for respond tumor microenvironment. In the present study, we first time fabricated a novel combination of oxidized sodium alginate-enveloped and 4T1 cancer cell membrane-coated up-conversion nanoparticles (4TUCNP@SMZ) were engineered for breast cancer-targeted therapy. The nanoparticles could be formed via electrostatic interaction, which showed excellent biocompatibility, increased cellular uptake with normal and cancer cells. The fabricated Alginate and 4T1-membrane functionalized up-conversion nanoparticles significantly benefitted to targeted delivery system for local breast cancer and prominently inhibit tumor development via chemo-dynamic therapy (CDT) and PDT. The greater susceptibility of tumor cells to oxidative stress may also result from the depletion of intracellular GSH by the 4TUCNP@SMZ nanoparticles. The in vitro cell models (4T1 and MCF-7), in vivo tumor model and histological observation demonstrated that 4TUCNP@SMZ nanoparticles effectively targeted to breast tumor microenvironment and inhibit breast cancer cells. This report suggested that multifunctional Alginate-functionalized nanoparticles are a versatile agent for phototherapy for breast cancer treatment.

Natural and polyanionic heparin polysaccharide functionalized upconversion nanoparticles for highly sensitive and selective ratiometric detection of pesticide

Pesticide contamination is a global concern, threatening human health and food safety. Herein, we developed heparin (HEP) functionalized upconversion nanoparticles (UCNPs)-based ratiometric nanosensor for the sensitive detection of 2,6-dichloro-4-nitroaniline (DCN) pesticide via inner filter effect. The strategy for HEP functionalization of UCNPs is based on adjusting the surface potentials of UCNPs with polyanionic HEP through the electrostatic interaction. UCNPs (NaYbF4:Gd/Y/Tm@NaYbF4@NaYF4) was designed with core-shell-shell structure and extra sensitizer layer for efficient and strong upconversion luminescence (UCL) in the range of UV to NIR. After incorporation of DCN, the upconverted UV emission of UCNPs-HEP ratiometric nanosensor was considerably quenched with the NIR UCL at 800 nm remaining unchanged as internal standard. The UCNPs-HEP ratiometric nanosensor can achieve outstandingly selective and sensitive detection of DCN at the wide linear range of 5–300 μM with a detection limit of 0.41 μM. The remarkable applicability of the UCNPs-HEP ratiometric nanosensor was verified in apple, cucumber and grapes samples. The developed UCNPs-HEP ratiometric nanosensor with excellent biocompatibility and water dispersion capability, is promising for convenient, selective and sensitive sensing of DCN towards food and aqueous samples.

Supersensitive detection of lincomycin with an ECL aptasensor based on the synergistic integration of gold-functionalized upconversion nanoparticles and thiolated 3,4,9,10-perylene tetracarboxylic acid.

In this work, the supersensitive and selective determination of lincomycin (Lin) was achieved using a novel electroluminescent (ECL) aptasensor based on the synergistic integration of gold functionalized upconversion nanoparticles (UCNPs) and thiolated 3,4,9,10-perylene tetracarboxylic acid (PTCA). The integration of two luminophores of UCNPs and PTCA combined the merits of the cathodoluminescence stability of UCNPs and the high quantum yield of PTCA, which significantly promoted the ECL signal and analytical performance of the proposed sensor. The introduction of gold nanoparticles in UCNPs can not only improve the conductivity and ECL performance of UCNPs but also cause them to easily integrate with thiolated PTCA (t-PTCA) via an Au-S bond. The ECL signal of UCNPs@Au/t-PTCA/GCE was almost twice as strong as that of t-PTCA/GCE and tenfold higher than that of UCNPs@Au/GCE. Because of the non-conductive protein of the Lin aptamer, the ECL intensity of apt/UCNPs@Au/t-PTCA/GCE noticeably decreased. In the presence of Lin, the aptamer was pulled down from the sensing interface, resulting in the recovery of the ECL intensity of the sensor. Under optimal conditions, our proposed sensor can quantify the concentration of Lin in the range from 1.0 × 10-15 to 1.0 × 10-7 M with a low detection limit of 2.4 × 10-16 M (S/N = 3), exhibiting high sensitivity and specificity for the determination of Lin.

Highly water-soluble and biocompatible hyaluronic acid functionalized upconversion nanoparticles as ratiometric nanoprobes for label-free detection of nitrofuran and doxorubicin

Antibiotic detection is crucial and challenging because the widespread consumption of antibiotics has shown extensive harmful effects on food, environment and human health. Here, we propose highly water-soluble and biocompatible hyaluronic acid (HYA) functionalized upconversion nanoparticles (UCNPs) for ratiometric detection of multiple antibiotics. The ultraviolet upconversion luminescence (UCL) from UCNPs was significantly quenched by nitrofurazone (NFZ)/nitrofurantoin (NFT), and blue UCL was quenched by doxorubicin (DOX), while red UCL remained unchanged for internal reference. The UCNPs-HYA nanoprobes exhibit excellently sensitive and selective NFZ, NFT and DOX detection in linear range of 2.5–100 μM, 2.5–80 μM, and 2.5–200 μM with the LOD at 0.28 μM (55 μg/kg), 0.20 μM (48 μg/kg) and 0.17 μM (97 μg/kg), respectively. The nanoprobes achieved detecting real samples of NFZ in lake water, liquid milk and chicken meat with satisfactory results, and UCL bioimaging of DOX in HeLa cells. The UCNPs-HYA ratiometric nanoprobes are promising for food samples detection and potential biosensing in the cellular environment.

An innovative solid-phase biosensor for rapid on-site detection of N-nitrosodimethylamine incorporating zein film and upconversion nanoparticles

Pickled frequently contains nitrosodimethylamine (NDMA), a mutagenic and carcinogenic substance that is dangerous for the general public's health. This study reports on the fabrication of a fluorescent biosensor using zein film and aptamer functionalized upconversion nanoparticles (UCNPs) for on-site monitoring of NDMA in meat. UCNPs were first prepared followed by aptamer binding and mixing with zein film, which was further conjugated with cDNA of dabcyl modified at 5′. The fluorescence resonance energy transfer (FRET) mechanism between the UCNPs and dabcyl was exploited. The fluorescence signals of the zein film recovered when NDMA was present because it was selectively collected by the particular aptamer and damaged the cDNA structure. The designed functionalized zein film was used for on-site and portable determination of NDMA with a lower limit of detection of 0.017 ng/mL, and possessed a satisfactory recovery ranging from 95.8% to 100.2% with no significant difference compared with the GC–MS method.

Fluorescence sensing platform for Cronobacter sakazakii based on the cationic metal-organic frameworks modified upconversion nanoparticles

Cronobacter sakazakii ( C. sakazakii ) is a serious threat to human health. Efficient detection of C. sakazakii is crucial for food safety and the control of foodborne diseases. Here, a fluorescence sensing platform for C. sakazakii was constructed with α-glucosidase and cationic metal-organic framework-functionalized upconversion nanoparticles (UCNPs@MOFs). The anionic α-glucosidase was inhibited by binding to cationic UCNPs@MOFs. The presence of C. sakazakii competitively combined with cationic UCNPs@MOFs and released α-glucosidase into the enzyme substrate solution (para-nitrophenyl-α- d -glucopyranoside) to recover activity, thus triggering fluorescence quenching of cationic UCNPs@MOFs through the product of 4-nitrophenol. In addition, α-glucosidase from C. sakazakii accelerated the formation of 4-nitrophenol, thus improving the fluorescence response of cationic UCNPs@MOFs. This strategy illustrates high sensitivity of the designed platform for C. sakazakii with a wide linear range (1.1×10 2 –1.1 × 10 8 cfu mL −1 ), a low limit of detection (3.2 × 10 1 cfu mL −1 ), and an acceptable recovery value (85.2–111.5%) in real samples (tap water, infant formula powder, and goat milk powder). These results confirm that the proposed fluorescence platform is an ideal candidate tool for the detection of C. sakazakii in microbiological diagnostics. • A highly sensitive and rapid fluorescence sensor for Cronobacter sakazakii was developed. • The advantage of sensor based on the excellent fluorescence of functionalized upconversion nanoparticles probes. • The quenching was mainly arising from the competitive combination of UCNPs probes and the anionic enzyme. • A broad linear range (1.1×10 2 -1.1 × 10 8 cfu mL −1 ) and a high sensitivity (3.2 × 10 1 cfu mL −1 ). • Cronobacter sakazakii in real samples (tap water, infant formula powder and goat milk powder) were successfully detected.

Autofluorescence free functionalized upconversion nanoparticles-based turn-on aptasensor for highly sensitive and selective sensing of antibiotics

Overuse of antibiotics such as enrofloxacin (ENR) can lead to their residues in environment and food and ultimately endanger human health. It's essential to build a rapid, sensitive and selective biosensor to detect the antibiotics residues. Herein, an autofluorescence free near-infrared (NIR) laser excited functionalized upconversion nanoparticles (UCNPs) based turn-on aptasensor for ENR detection is presented. Efficient Fӧrster resonance energy transfer (FRET) from aptamer modified core-shell UCNPs with highly enhanced upconversion (UC) luminescence to MnO2 nanosheets occurs. Based on the UC luminescence recovery of the UCNPs-MnO2 aptasensor caused by ENR, this luminescence turn-on UCNPs-MnO2 aptasensor shows highly sensitive and selective detection of ENR in linear range of 0.5–3000 ng mL−1 with a limit of detection (LOD) at 0.0642 ng mL−1 in diluted commercially available milk solution without pretreatment. The proposed aptasensor demonstrates high sensitivity, excellent selectivity, no background autofluorescence for high signal-to-noise ratio and low LOD for the fast detection of antibiotics residue, holding great potential for environmental and food safety detection of antibiotics.

Bipyridine-mediated fluorescence charge transfer process based on copper ion grafted upconversion nanoparticle platform for ciprofloxacin sensing in aquatic products

In this work, an ultrasensitive ciprofloxacin (CIP) detection strategy has been established based on copper (Cu2+) ions-induced strong charge transfer in poly acrylic acid (PAA) functionalized upconversion nanoparticles (UCNPs)/2,2-bipyridine (bipy) system. The positively charged Cu2+ ions electrostatically adhere to the surface of the PAA-UCNPs and deactivate the fluorescence via a charge transfer process. The bipy in this hybrid system controls the aggregation by chelating in proximity to the Cu2+ center. Due to the strong affinity between pyridone oxygen and carboxy oxygen, CIP coordinates in high stoichiometry with the bipy-Cu complex as compared to the PAA-UCNPs, causing the trapped fluorescence to be released in an amount equivalent to the target concentration. Under the optimum assay conditions, a good calibration plot (0.05–1000 ng/mL) was acquired with a detection limit of 0.13 ng/mL. The satisfactory recoveries (85.93–96.87%) for real prawn and fish samples were further validated by enzyme-linked immunoassays (P > 0.05).

Upconversion photoluminescent Yb(Ⅲ)/Er(Ⅲ) doped nanoparticles interact with AuNPs for detection of Cr (Ⅲ) and sodium tripolyphosphate based on FRET

In this study, we designed a dual functional nanosensor for detecting Cr (Ⅲ) and sodium tripolyphosphate (STPP) based on fluorescence resonance energy transfer (FRET) mechanism between amine functionalized upconversion nanoparticles (UCNPs) and gold nanoparticles (AuNPs), where UCNPs serve as energy donor and aggregated AuNPs as energy acceptor. UCNPs have red emission at 654 nm under 980 nm laser excitation and monodispersed AuNPs show strong absorbance around 520 nm. Monodispersed AuNPs form aggregate easily in the presence of Cr (Ⅲ), which leads to the red-shift of the absorption of AuNPs and quenches the red emission of UCNPs. In the presence of STPP, the formation of Cr (Ⅲ)-STPP complex weakens the aggregation extent of AuNPs and the red emission of UCNPs increases. Therefore, the content of Cr (Ⅲ) and STPP could be detected in wide range according to the linear variety of fluorescence intensities of UCNPs at 654 nm with the detection limits of 0.44 μM and 0.54 μM for detecting Cr (Ⅲ) and STPP, respectively. In addition, due to the high selectivity and accuracy for the detection of STPP, this nanosensor can be utilized to determine STPP in green tea drink and drinking water.

Functionalized Upconversion Nanoparticles Based Turn-On Aptasensor for Highly Sensitive and Selective Detection of Antibiotics

Functionalized Upconversion Nanoparticles Based Turn-On Aptasensor for Highly Sensitive and Selective Detection of Antibiotics

Photo- and pH-responsive drug delivery nanocomposite based on o-nitrobenzyl functionalized upconversion nanoparticles

A near infrared (NIR) and pH dual-responsive nanocomposite, composed of a lanthanide-doped upconversion nanoparticle (UCNP) core and a transformable poly-o-nitrobenzyl shell, was prepared by the distillation precipitation polymerization and template method. The poly-o-nitrobenzyl shell can undergo hydrophobic-hydrophilic transformation upon an irradiation of 980 nm, rendering the capability of NIR-activated drug release. The anticancer drug, doxorubicin (DOX), can be loaded into these nanocomposites with a loading efficiency of 7.23 wt%. Furthermore, pH responsiveness caused by the hydrogen bond and charge interactions between DOX and nanocapsules can trigger the release of drugs at low pH. Under visible light irradiation and neutral (pH 7.4) conditions, the cumulative release of DOX was only 8.35% after 300 min, while it reached 59.5% under the synergistic effect of NIR irradiation and acidic conditions. The Baker-Lonsdale model was used to describe the drug release kinetics of this system, and the diffusion coefficient (3DCs/r02C0) and R2 under different conditions were determined to be 4.15 × 10−6 and 0.98 (pH 7.4 and visible light), 2.64 × 10−5 and 0.99 (NIR light), 3.26 × 10−5 and 0.97 (pH 4.5 and visible light), 2.59 × 10−4 and 0.99 (pH 4.5 and NIR light), respectively. This controlled release feature makes the nanocomposite a promising therapeutic agent for treating diseases.

Dual-mode of magnetic assisted Au@Ag SERS tags and cationic conjugated UCNPs for qualitative and quantitative analysis of multiple foodborne pathogens

A new dual-mode optical-nano-sensor was designed to effectively isolate, discriminate and inactivate multiple foodborne pathogens through the combined effort of magnetic SERS tags and functionalized upconversion nanoparticles. The positively charged quaternary ammonium salt (QAS) functionalized upconversion nanoparticles (UCNPs@QAS) were used as a fluorescent probe for the analysis of total bacteria count and inactivation of bacteria, and 4-mercaptophenylboronic acid (4-MPBA) modified magnetic Au@Ag NPs (MAu@Ag@MPBA) were applied as a SERS tag for isolation and classification of bacteria. Various types of bacteria can be captured by UCNPs@QAS through electrostatic interactions and by MAu@Ag@MPBA via the reversible recognition interaction of a boronic acid group of 4-MPBA and peptidoglycan from bacteria simultaneously. And then the formed complex structural assemblies were isolated rapidly from the sample matrix by employing the magnetic force for fast and simple dual-model optical detection. In addition, the novel biosensor was successfully applied to discriminate and detect Escherichia coli, Salmonella, Staphylococcus aureus, and their mixture in contaminated milk with the classification rate of 97.2–100 %. The proposed multi-functional nano-platform was able to sensitively capture, detect, and kill foodborne pathogens in a complex system, which could be deployed successfully towards the monitoring of food safety.

Functionalized upconversion nanoparticles: New strategy towards FRET-based luminescence bio-sensing

• Current advancement in functionalized UCNPs for FRET-based bio-sensing. • Discussed impact of surface functionalization of UCNPs to enhanced the loading/detection limit. • Importance of UCNPs in optical bio-probe/detection. • Role of co-dopants is discussed to enhance the emission efficiency. • Future challenges and improvement of UCNPs based luminescent biosensing are proposed. Functionalized upconversion nanoparticles (UCNPs) have perceived massive success in the area of bio-photonic applications. UCNPs are extensively used as a successful emitter in terms of unique luminescent properties, minimum auto-fluorescence, high chemical &thermal photo-stability, deep tissue penetration, excellent biocompatibility, and low toxicity. The relatively high emission efficiency of the UCNPs creates them greatly attractive and encouraging materials for widespread usages in biomedical and clinical sciences. High color purity, easy surface functionalization, and good colloidal stability are the advantageous features of the UCNPs. Widespread efforts are dedicated to emerging operational approaches for the preparation and surface functionalization/engineering of luminescent UCNPs of varying morphologies, simplifying the advancement of this auspicious material for fluorescent resonance energy transfer (FRET) based luminescence biosensing applications. We widely review the recent advancement of UCNPs in optical bio-sensing applications, with importance on the synthesis, surface functionalization, emission efficiency enhancement factors, and their successful use in the detection of pathogenic diseases, free radicals, and biomarkers. A detailed overview of functionalization techniques for UCNPs has also been presented, with a special emphasis on organic ligands-functionalization, doping, and methods of biological species-encapsulation/conjugation. Based on the current scientific achievements, traditional optical applications such as bio-sensing, immunosensing, biomarkers are now added. Finally, as a roadmap scheme for study in this area, future trends and recent problems are emphasized.

Uptake of Polyelectrolyte Functionalized Upconversion Nanoparticles by Tau-Aggregated Neuron Cells.

Tauopathy is the aggregation phenomenon of tau proteins and associated with neurodegenerative diseases. It metastasizes via the transfer of tau aggregates to adjacent neuron cells; however, the mechanism has not yet been fully understood. Moreover, if the materials used for designing drug delivery system to treat such neurodegenerative diseases do not undergo biodegradation or exocytosis but remains in cells or tissues, they raise concerns about their possible negative impacts. In this study, the uptake and delivery mechanisms of nano-sized carriers in tau aggregated neuron cells were investigated employing polyelectrolyte-functionalized upconversion nanoparticles (UCNPs) of diameter ~100 nm. Investigation through bioimaging was carried out by irradiating the particles with near-infrared light. Here, forskolin and okadaic acid were employed to induce tau aggregation into healthy neuron cells. It was noticed that the tau-aggregated neuron cells, when treated with relatively large sized UCNPs, showed uptake efficiency similar to that of normal neuron cells however their intracellular transport and exocytosis were impacted, and most of the carriers remained accumulated around lysosome. This demonstrates that metastasis mechanisms of tauopathy can get influenced by the size of carriers and are to be considered during their pharmacokinetic studies which is often not addressed in many drug delivery studies.

Recent advances in functionalized upconversion nanoparticles for light-activated tumor therapy.

Upconversion nanoparticles (UCNPs) are a class of optical nanocrystals doped with lanthanide ions that offer great promise for applications in controllable tumor therapy. In recent years, UCNPs have become an important tool for studying the treatment of various malignant and nonmalignant cutaneous diseases. UCNPs convert near-infrared (NIR) radiation into shorter-wavelength visible and ultraviolet (UV) radiation, which is much better than conventional UV activated tumor therapy as strong UV-light can be damaging to healthy surrounding tissue. Moreover, UV light generally does not penetrate deeply into the skin, an issue that UCNPs can now address. However, the current studies are still in the early stage of research, with a long way to go before clinical implementation. In this paper, we systematically analysed recent advances in light-activated tumor therapy using functionalized UCNPs. We summarized the purpose and mechanism of UCNP-based photodynamic therapy (PDT), gene therapy, immunotherapy, chemo-therapy and integrated therapy. We believe the creation of functional materials based on UCNPs will offer superior performance and enable innovative applications, increasing the scope and opportunities for cancer therapy in the future.

Functionalized Upconversion Nanoparticles for Disassembly of β‑Amyloid Aggregation with Near-Infrared Excitation

Functionalized Upconversion Nanoparticles for Disassembly of β‑Amyloid Aggregation with Near-Infrared Excitation

Upconversion Fluorescent Nanoprobe for Highly Sensitive In Vivo Cell Tracking.

Near-infrared (NIR)-to-visible upconversion nanomaterials (UCNPs) used as biomedical nanoprobes have considerable advantages over the traditional used "downconversion" fluorescent dyes. Functionalized upconversion nanoparticles (UCNPs) represent high sensitivity and great biocompatibility. Cells labeled with these UCNPs can be tracked for long term in vivo. Here we describe UCNP-PEG-ARG for highly sensitive in vivo cell tracking.

Functionalized Upconversion Nanoparticles for Targeted Labelling of Bladder Cancer Cells.

Bladder cancer is the ninth most common cancer worldwide. Due to a high risk of recurrence and progression of bladder cancer, every patient needs long-term surveillance, which includes regular cystoscopy, sometimes followed by a biopsy of suspicious lesions or resections of recurring tumours. This study addresses the development of novel biohybrid nanocomplexes representing upconversion nanoparticles (UCNP) coupled to antibodies for photoluminescent (PL) detection of bladder cancer cells. Carrying specific antibodies, these nanoconjugates selectively bind to urothelial carcinoma cells and make them visible by emitting visible PL upon excitation with deeply penetrating near-infrared light. UCNP were coated with a silica layer and linked to anti-Glypican-1 antibody MIL38 via silica-specific solid-binding peptide. Conjugates have been shown to specifically attach to urothelial carcinoma cells with high expression of Glypican-1. This result highlights the potential of produced conjugates and conjugation technology for further studies of their application in the tumour detection and fluorescence-guided resection.

Preparation and Evaluation of Upconversion Nanoparticles Based miRNA Delivery Carrier in Colon Cancer Mice Model.

Therapeutic efficacy of solid tumor is often severely hampered by poor penetration of therapeutics into diseased tissues and lack of tumor targeting. In this study, the functionalized upconversion nanoparticles (UCNP)-based delivery vector targeting cancer cells was developed. Firstly, NaYF₄:Yb/Tm (UCNP) was prepared with the solvothermal method for the uniform nanoparticle size and brilliant lattice structure. The SiO₂ coated UCNP was demonstrated a high upconversion emission and good monodispersity, which was coupled with polyetherimide (PEI) and miR-145 vector. Then, it was further functionalized via hyaluronic acid (HA) (UCNP/PEI/HA Nanocomplex, UCNPs) coating for the targeted delivery and improved biocompatibility. The UCNPs/miR-145 displays an excellent biocompatibility, a high level of cellular uptake and miR-145 expression, which results in a significant cell cycle arrest in G1, and induces CCND1, CDK6 and CCNE2 proteins downregulation. In vivo, the HA-coated UCNPs were enriched at the tumor site by targeting and retention effects, which resulted in a significant inhibition of tumor growth. Histological experiments demonstrated that UCNPs did not show significant toxicity in mice colon cancer model. Taken together, a UCNPs-based delivery platform was successfully constructed and used for miRNA target delivery, which provided a new method and idea for bioengineering and nanotechnology-based tumor therapy.

Surfactant-Sensitized Covalent Organic Frameworks-Functionalized Lanthanide-Doped Nanocrystals: An Ultrasensitive Sensing Platform for Perfluorooctane Sulfonate.

Perfluorooctane sulfonate (PFOS) known as a persistent organic pollutant has been attracting great interests due to its potential ecotoxicity. An approach capable of sensing ultra-trace PFOS is in urgent demand. Here, we developed an approach for highly sensitive sensing PFOS using surfactant-sensitized covalent organic frameworks (COFs)-functionalized upconversion nanoparticles (UCNPs) as a fluorescent probe. COFs-functionalized UCNPs (UCNPs@COFs) were obtained by solvothermal growth of 1,3,5-triformylbenzene and 1,4-phenylenediamine on the surface of UCNPs. COF’s layer on the surface of UCNPs not only provides recognition sites for PFOS but also improves the fluorescence quantum yields from 2.15 to 5.12%. Trace PFOS can quench the fluorescence emission of UCNPs@COFs at 550 nm due to the high electronegativity of PFOS. Moreover, the fluorescence quenching response can be significantly strengthened in the presence of a surfactant, which causes more sensitivity. The fluorescence quenching degrees (F0 – F) of the system are linear with the concentration of PFOS in the range of 1.8 × 10–13 to 1.8 × 10–8 M. The present sensor can sensitively and selectively detect PFOS in tap water and food packing with the limit of detection down to 0.15 pM (signal-to-noise ratio = 3), which is comparable to that of the liquid chromatography–mass spectrometry technique. The proposed approach realized a simple, fast, sensitive, and selective sensing PFOS, showing potential applications in various fields.