One-pot hydrothermal synthesis of fluorophore-modified cerium oxide nanoparticles.

Cerium oxide nanoparticles (CeO2 NPs), which have powerful antioxidant properties, are promising nanomaterials for the treatment of diseases associated with oxidative stress. The well-developed surface of CeO2 NPs makes them promising for use as a multifunctional system for various biomedical applications. This work demonstrates a simple approach that allows the direct formation of a molecular fluorophore on the surface of CeO2 NPs using a simple one-pot hydrothermal synthesis. Thus, we were able to synthesize CeO2 NPs of ultra-small size ∼2 nm with a narrow distribution, highly stable fluorescence, and a quantum yield of ∼62%. UV-visible transmission studies revealed that the resulting CeO2 NPs exhibited fast autogenerative catalytic reduction. In vitro results showed high biocompatibility of CeO2 NPs; their internalization occurs mainly in the region of cell nuclei. Thus, the resulting NPs have the necessary parameters and can be successfully used in biovisualization and therapy.

Effect of photoconversion conditions on the spectral and cytotoxic properties of photoconvertible fluorescent polymer markers.

Fluorescence labeling of cells is a versatile tool used to study cell behavior, which is of significant importance in biomedical sciences. Fluorescent photoconvertible markers based on polymer microcapsules have been recently considered as efficient and perspective ones for long-term tracking of individual cells. However, the dependence of photoconversion conditions on the polymeric capsule structure is still not sufficiently clear. Here, we have studied the structural and spectral properties of fluorescent photoconvertible polymeric microcapsules doped with Rhodamine B and irradiated using a pulsed laser in various regimes, and shown the dependence between the photoconversion degree and laser irradiation intensity. The effect of microcapsule composition on the photoconversion process was studied by monitoring structural changes in the initial and photoconverted microcapsules using X-ray diffraction analysis with synchrotron radiation source, and Fourier transform infrared, Raman and fluorescence spectroscopy. We demonstrated good biocompatibility of free-administered initial and photoconverted microcapsules through long-term monitoring of the RAW 264.7 monocyte/macrophage cells with unchanged viability. These data open new perspectives for using the developed markers as safe and precise cell labels with switchable fluorescent properties.

Labeling and Tracking of Individual Human Mesenchymal Stromal Cells Using Photoconvertible Fluorescent Microcapsules.

The behavior and migration of human mesenchymal stromal cells (hMSCs) are focal points of research in the biomedical field. One of the major aspects is potential therapy using hMCS, but at present, the safety of their use is still controversial owing to limited data on changes that occur with hMSCs in the long term. Fluorescent photoconvertible proteins are intensively used today as "gold standard" to mark the individual cells and study single-cell interactions, migration processes, and the formation of pure lines. A crucial disadvantage of this method is the need for genetic modification of the primary culture, which casts doubt on the possibility of exploring the resulting clones in personalized medicine. Here we present a new approach for labeling and tracking hMSCs without genetic modification based on the application of cell-internalizable photoconvertible polyelectrolyte microcapsules (size: 2.6 ± 0.5 µm). These capsules were loaded with rhodamine B, and after thermal treatment, exhibited fluorescent photoconversion properties. Photoconvertible capsules demonstrated low cytotoxicity, did not affect the immunophenotype of the hMSCs, and maintained a high level of fluorescent signal for at least seven days. The developed approach was tested for cell tracking for four days and made it possible to trace the destiny of daughter cells without the need for additional labeling.

Luminescent alloyed quantum dots for turn-off enzyme-based assay.

A new bioanalytical labeling system based on alloyed quantum dots' (QDs) photoluminescence quenching caused by an enzymatic reaction has been developed and tested for the first time. The catalytic role of the enzyme provides high sensitivity and the possibility of varying detecting time to improve assay sensitivity. Alloyed luminescent QDs were chosen in view of their small size (5-7 nm) and the high sensitivity of their optical properties to physicochemical interactions. Here, we described the synthesis of alloyed luminescent QDs and demonstrated the possibility of using them as a luminescent turn-off substrate for enzymatic assay. Synthesized alloyed QDs were found to be a sensitive turn-off substrate for glucose oxidase in homogeneous and heterogeneous assay models. CdZnSeS and CdZnSeS/ZnS QDs covered with dihydrolipoic acid and 2-mercaptoethanol were tested. A glucose oxidase limit of detection of 6.6 nM for the heterogenous high-throughput model assay was reached.

Nets of biotin-derived gold nanoparticles as a label for the C-reactive protein immunoassay.

This study presents a promising approach for the one-pot generation of the biotin-derived gold nanoparticles (GNPs@biotin). We report a direct method for the reduction of tetrachloroauric acid with biotin and generation of the labels due to nets formed via biotin-streptavidin interactions. The synthesized GNPs@biotin have a characteristic plasmon maximum, excellent colloidal stability, and streptavidin coupling efficiency. The size of the GNPs@biotin:streptavidin nets and the efficiency of interaction with specific antibodies can be easily customized by the component concentrations and time of their interaction. Moreover, the proposed labels require no additional reagents or manipulations for the synthesis, separation, or purification. The developed labels were applied for the detection of the model antigen of C-reactive protein (CRP) as a major inflammation biomarker. The assembling labels demonstrated a competitive advantage limit of CRP detection (LOD) of 1.2 ng/mL and a limit of quantification (LOQ) of 3.9 ng/mL in human plasma comparable to classical immunoassays. Moreover, the proposed approach is universal and can be potentially applied for the quantitative determination of other biomarkers in a variety of immunoassays in a combination with specific biotinylated antibodies.

Gold based nano-photonic approach for point-of-care detection of circulating long non-coding RNAs.

Development of a rapid, sensitive and easy to use point of care assay for detection of circulating long non-coding RNAs (lncRNAs) is of great importance. These biomolecules possess the ability to regulate vital cellular processes and act as biomarkers for various human non-communicable diseases. The present work aimed to develop a simplified and reliable cytometric fluorescence-based approach for precise recognition of circulating lncRNAs in a given sample using biotinylated uracil-modified oligonucleotide tethered AlexaFluor488-labeled streptavidin gold colloidal (BiO-StrAG) nano-conjugates. The fluorophores in close proximity to the gold nanoparticles result in quenching of fluorescence; however, specific recognition of target lncRNAs increases this distance which causes plasmonic enhancement of fluorescence. As per the flow cytometry and fluorometry investigations, the developed methodology provides a precise and sensitive approach for detection of the target lncRNAs (up to 5 nM in any given sample). With advantages of high selectivity and feasibility, our strategy offers great potential of being developed as a promising tool for interrogating aberrant regulation of lncRNAs functions, especially indicated in various diseased states.

Molecularly imprinted polyaniline for detection of horseradish peroxidase.

A new facile and fast approach to the synthesis of polyaniline (PANi) molecularly imprinted polymers (MIPs) based on aniline oxidative chemical polymerization was proposed for protein recognition. For the first time, a surface imprinting strategy was implemented for the synthesis of PANi MIPs on the inner surface of soft glass polycapillaries (PC) with a large (2237) number of individual microcapillaries. Two different PANi layers-(i) PANi film and (ii) protein imprinted PANi nanowires-were synthesized sequentially. Uniform and highly stable PANi film was synthesized by oxidative polymerization at pH< 1. The synthesis of PANi MIPs on the PANi film pre-coated surface improved the reproducibility of PANi MIP formation. PANi MIP nanowires were synthesized at "mild" conditions (pH > 4.5) to preserve the protein template activity. The binding of horseradish peroxidase (HRP) molecules on the PANi MIP selective sites was confirmed by photometry (TMB chromogenic reaction), SEM images, and FTIR spectroscopy. The developed PANi MIPs enable HRP determination with a limit of detection (LOD) as low as 1.00 and 0.07 ng mL-1 on the glass slips and PC, respectively. The PANi MIPs are characterized by high stability; they are reversible and selective to HRP. The proposed approach allows PANi MIPs to be obtained for proteins on different supports and to create new materials for separation and sensing. Graphical abstract.

Fluorescent AgInS/ZnS quantum dots microplate and lateral flow immunoassays for folic acid determination in juice samples.

A noninstrumental rapid test for folic acid (FA) detection with visual results evaluation utilizing bright water-stable AgInS/ZnS (AIS/ZnS) quantum dots (QDs) is reported . AIS/ZnS QDs are hydrophilic photostable nanocrystals with size < 7 nm and emission in the visible range. They were synthesized directly in the water phase by a simple method compared to the synthesis of other QDs and conjugated with monoclonal antibodies specific for FA via ligand carboxyl groups. The conjugate was used for the development of instrumental qualitative and rapid quantitative FA detection methods. The competitive fluorescent microplate immunosorbent assay provided a limit of detection of 0.1 ng/mL FA and half maximal inhibitory concentration (IC50) of 24 ng/mL FA. The analytical signal was measured at ʎex = 410 nm and ʎem=590 nm. The proposed method showed no cross-reaction with other group B vitamins. For FA screening in juice samples, the lateral flow immunoassay was developed with a visual cutoff level of 3 µg/mL. In our perception, the developed methods are convenient for proving the perception of the AIS/ZnS QDs application as a luminescent label for immunoassay and are effective for FA detection. Graphical abstract.

Mapping the Mitochondrial Regulation of Epigenetic Modifications in Association With Carcinogenic and Noncarcinogenic Polycyclic Aromatic Hydrocarbon Exposure.

Polycyclic aromatic hydrocarbons (PAHs) refer to a ubiquitous group of anthropogenic air pollutants that are generated through incomplete carbon combustion. Although the immunotoxic nature of PAHs has been previously reported, the underlying molecular mechanisms of this effect are not fully understood. In the present study, we investigated the mitochondrial-mediated epigenetic regulation of 2 PAHs, carcinogenic (benzo[a]pyrene; BaP) and noncarcinogenic (anthracene [ANT]), in peripheral lymphocytes. While ANT exposure triggered mitochondrial oxidative damage, no appreciable epigenetic modifications were observed. On the other hand, exposure to BaP perturbed the mitochondrial redox machinery and initiated cascade of epigenetic modifications. Cells exposed to BaP showed prominent changes in the expression of mitochondrial microRNAs (miR-24, miR-34a, miR-150, and miR-155) and their respective gene targets (NF-κß, MYC, and p53). The exposure of BaP also caused significant alterations in the expression of epigenetic modifiers (DNMT1, HDAC1, HDAC7, KDM3a, EZH2, and P300) and hypomethylation within nuclear and mitochondrial DNA. This further induced methylation of histone tails, which play a crucial role in the regulation of chromatin structure. Overall, our study provides novel mechanistic insights into the mitochondrial regulation of epigenetic modifications in association with PAH-induced immunotoxicity.

Carbon dot aggregates as an alternative to gold nanoparticles for the laser-induced opening of microchamber arrays.

Carbon dots (CDs) are usually used as an alternative to other fluorescent nanoparticles. Apart from fluorescence, CDs also have other important properties for use in composite materials, first of all their ability to absorb light energy and convert it into heat. In our work, for the first time, CDs have been proposed as an alternative to gold nanostructures for harvesting light energy, which results in the opening of polymer-based containers with biologically active compounds. In this paper, we propose a method for the synthesis of polylactic acid microchamber arrays with embedded CDs. A comparative analysis was made of the damage to microchambers functionalized with gold nanorods and with CD aggregates, depending on the wavelength and power of the laser used. The release of fluorescent cargo from the microchamber arrays with CD aggregates under laser exposure was demonstrated.

Sample pretreatment and SERS-based detection of ceftriaxone in urine.

The aim of the work is the development of the procedure for ceftriaxone (antibiotic drug of cephalosporin class) detection in urine using surface-enhanced Raman spectroscopy (SERS). Hydroxylamine stabilized silver nanoparticles were used as SERS-active material. Additional urine pretreatment steps were developed in order to eliminate the influence of creatinine on the ceftriaxone SERS signal. These steps include adjusting of the sample pH to alkaline value (pH 13) and purification of the sample using silica gel column chromatography. Alkali pH increases SERS signal of ceftriaxone, while silica gel separates the analyte from creatinine-the main admixture in urine which provides inappropriate SERS signal background. Additionally, it was found that total protein content up to 0.2 mg/mL (upper level for urine of healthy person) and pH deviation of initial urine do not influence on SERS signal of ceftriaxone. The proposed detection procedure enables fast (~ 10 min) determination of ceftriaxone in artificially spiked urine samples within 5 to 500 µg/mL range of concentrations which matches the range of the drug concentrations in urine after injection of therapeutically required dosages. Limits of detection (3σ) and quantification (10σ) were found to be 0.4 and 2.0 µg/mL, correspondingly. Graphical abstract Application of urine pretreatment enables the purification of target analyte from intrinsic urine components and improves SERS-based detection of ceftriaxone (antibiotic drug).

Fluorescently labelled multiplex lateral flow immunoassay based on cadmium-free quantum dots.

A sensitive tool for simultaneous qualitative detection of two mycotoxins based on use of non-cadmium quantum dots (QDs) is presented for the first time. QDs have proven themselves as promising fluorescent labels for biolabeling and chemical analysis. With an increasing global tendency to regulate and limit the use of hazardous elements, indium phosphide (InP) QDs are highlighted as environmentally-friendly alternatives to the highly efficient and well-studied, but potentially toxic Cd- and Pb-based QDs. Here, we developed water-soluble InP QDs-based fluorescent nanostructures. They consisted of core/shell InP/ZnS QDs enrobed in a silica shell that allowed the water solubility (QD@SiO2). Then we applied the QD@SiO2 as novel, silica shell-encapsulated fluorescent labels in immunoassays for rapid multiplexed screening. Two mycotoxins, zearalenone and deoxynivalenol, were simultaneously detected in maize and wheat, since the two QD@SiO2 labelled conjugates emit at two different, individually detectable wavelengths. The cutoff values for the simultaneous determination were 50 and 500µgkg-1 for zearalenone and deoxynivalenol, respectively, in both maize and wheat. Liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) was used to confirm the result.

SERS detection of ceftriaxone and sulfadimethoxine using copper nanoparticles temporally protected by porous calcium carbonate.

The authors describe a new composite based on SERS-active copper nanoparticles (CuNPs; 10 ± 2 nm) incorporated into calcium carbonate microspheres (CaCO3-CuNPs; 3.4 ± 0.3 µm). The CaCO3 coating acts as a temporal protector of CuNPs against oxidation. Incorporated CuNPs have significantly improved stability during storage and a month-long shelf lifetime. The composite was used for SERS detection of rhodamine 6G and two antibacterial drugs (ceftriaxone and sulfadimethoxine). Two analytical formats, one with and one without solid phase extraction, are introduced to demonstrate the flexibility of the method. Both formats imply the dissolution of CaCO3 matrix before SERS analysis to release CuNP used as SERS substrate. The study of the influence of pH value and acid nature on the SERS signal demonstrated that HCl is the most efficient candidate to release the CuNPs. Sensitivity (expressed as LOD) is shown to be improved by more than one order when solid phase extraction is used. The average SERS enhancement factor is 10^7 which makes the material efficiency comparable to the one of silver nanoparticles. The LOD (<5 µM), precision (RSDs between 20 and 24% at LOD levels), and trueness (apparent recoveries 84-113%) for the two antibiotics (ceftriaxone and sulfadimethoxine) make the method quite useful for quantitative analysis and therapeutic drug monitoring at physiologically relevant concentrations. Graphical abstract A composite with temporally stable copper nanoparticles was synthesized, studied, and used for SERS detection of two antibacterial drugs. The analytical efficiency of the composite was found appropriate for quantitative analysis due to Raman enhancement comparable with silver nanostructures.

SERS as a tool for determination of structurally related compounds: The case of sulfanilamide class antibiotics.

Analysis of real objects based on surface-enhanced Raman spectroscopy (SERS) often utilizes new SERS substrates and/or complex analysis procedures, and they are optimized for only the determination of a single analyte. Moreover, analysis simplicity and selectivity are often sacrificed for maximum (sometimes unnecessary) sensitivity. Consequently, this trend limits the versatility of SERS analysis and complicates its practical implementation. Thus, we have developed a universal, but simple SERS assay suitable for the determination of structurally related antibiotics (five representatives of the sulfanilamide class) in complex objects (human urine and saliva). The assay involves only mixing of acidified analyzed solution with co-activating agent (polydiallyldimethylammonium chloride - PDDA) and SERS substrate (standard colloidal silver nanoparticles). Acidification promotes the generation of SERS spectra with maximum similarity and intensity, which is explained by the favorable enhancement of the protonated sulfanilamide moiety (a structurally similar part of the studied antibiotics) as a result of its strong electrostatic interaction with the SERS-active surface. Meanwhile, the addition of PDDA improves analysis selectivity by reducing background signal from body fluids, enabling to simplify sample pretreatment (dilution for urine; mucin removal and dilution for saliva). Therefore, the assay allows for rapid (≤10 min), precise, and accurate class-specific determination of sulfanilamides within concentration ranges suitable for non-invasive therapeutic drug monitoring in urine (40-600 µM) and saliva (10-30 µM). We also believe that thorough investigation of structurally related analytes and accompanying effects (e.g., high spectral similarity) is a promising direction to improve the understanding of SERS in general and expand its capabilities as an analytical tool.

Visualization of 2D and 3D Tissue Models via Size-Selected Aqueous AgInS/ZnS Quantum Dots.

Three-dimensional (3D) spheroid cell cultures of fibroblast (L929) and tumor mammary mouse (4T1) were chosen as in vitro tissue models for tissue imaging of ternary AgInS/ZnS fraction quantum dots (QDs). We showed that the tissue-mimetic morphology of cell spheroids through well-developed cell-cell and cell-matrix interactions and distinct diffusion/transport characteristics makes it possible to predict the effect of ternary AgInS/ZnS fraction QDs on the vital activity of cells while simultaneously comparing with classical two-dimensional (2D) cell cultures. The AgInS/ZnS fractions, emitting in a wide spectral range from 635 to 535 nm with a mean size from ∼3.1 ± 0.8 to ∼1.8 ± 0.4 nm and a long photoluminescence lifetime, were separated from the initial QD ensemble by using antisolvent-induced precipitation. For ternary AgInS/ZnS fraction QDs, the absence of toxicity at different QD concentrations was demonstrated on 2D and 3D cell structures. QDs show a robust correlation between numerous factors: their sizes in biological fluids over time, penetration capabilities into 2D and 3D cell structures, and selectivity with respect to penetration into cancerous and healthy cell spheroids. A reproducible protocol for the preparation of QDs along with their unique biological properties allows us to consider ternary AgInS/ZnS fraction QDs as attractive fluorescent contrast agents for tissue imaging.

Multiple dyes applications for fluorescent convertible polymer capsules as macrophages tracking labels.

Tracing individual cell pathways among the whole population is crucial for understanding their behavior, cell communication, migration dynamics, and fate. Optical labeling is one approach for tracing individual cells, but it typically requires genetic modification to induce the generation of photoconvertible proteins. Nevertheless, this approach has limitations and is not applicable to certain cell types. For instance, genetic modification often leads to the death of macrophages. This study aims to develop an alternative method for labeling macrophages by utilizing photoconvertible micron-sized capsules capable of easy internalization and prolonged retention within cells. Thermal treatment in a polyvinyl alcohol gel medium is employed for the scalable synthesis of capsules with a wide range of fluorescent dyes, including rhodamine 6G, pyronin B, fluorescein, acridine yellow, acridine orange, thiazine red, and previously reported rhodamine B. The fluorescence brightness, photostability, and photoconversion ability of the capsules are evaluated using confocal laser scanning microscopy. Viability, uptake, mobility, and photoconversion studies are conducted on RAW 264.7 and bone marrow-derived macrophages, serving as model cell lines. The production yield of the capsules is increased due to the use of polyvinyl alcohol gel, eliminating the need for conventional filtration steps. Capsules entrapping rhodamine B and rhodamine 6G meet all requirements for intracellular use in individual cell tracking. Mass spectrometry analysis reveals a sequence of deethylation steps that result in blue shifts in the dye spectra upon irradiation. Cellular studies on macrophages demonstrate robust uptake of the capsules. The capsules exhibit minimal cytotoxicity and have a negligible impact on cell motility. The successful photoconversion of RhB-containing capsules within cells highlights their potential as alternatives to photoconvertible proteins for individual cell labeling, with promising applications in personalized medicine.

Anodic-stripping voltammetric immunoassay for ultrasensitive detection of low-abundance proteins using quantum dot aggregated hollow microspheres.

A new anodic-stripping voltammetric immunoassay protocol for detection of IgG1, as a model protein, was designed by using CdS quantum dot (QD) layer-by-layer assembled hollow microspheres (QDHMS) as molecular tags. Initially, monoclonal anti-human IgG1 specific antibodies were anchored on amorphous magnetic beads preferably selective to capture F(ab) of IgG1 analyte from the sample. For detection, monoclonal anti-human IgG1 (F(c)-specific) antibodies were covalently coupled to the synthesized QDHMS. In a sandwich-type immunoassay format, subsequent anodic-stripping voltammetric detection of cadmium released under acidic conditions from the coupled QDs was conducted at an in situ prepared mercury film electrode. The immunoassay combines highly efficient magnetic separation with signal amplification by the multilayered QD labels. The dynamic concentration range spanned from 1.0 fg mL(-1) to 1.0 µg mL(-1) of IgG1 with a detection limit of 0.1 fg mL(-1). The electrochemical immunoassay showed good reproducibility, selectivity, and stability. The analysis of clinical serum specimens revealed good accordance with the results obtained by an enzyme-linked immunosorbent assay method. The new immunoassay is promising for enzyme-free, and cost-effective analysis of low-abundance biomarkers.

A systematic assessment of the variability of matrix effects in LC-MS/MS analysis of ergot alkaloids in cereals and evaluation of method robustness.

The study presents for the first time a systematic investigation of matrix effects in the LC-MS/MS analysis of ergot alkaloids in cereals. In order to assure the accuracy of the results, several approaches to minimize/eliminate matrix effects were investigated including variation of ionization techniques, chromatography and sample preparation on different grain types and grain varieties. It was revealed that the use of UPLC and careful choice of sample preparation might reduce signal suppression/enhancement. In general, ergometrine was found to be the most susceptible among the ergot alkaloids studied, but none of the used approaches suggested a total elimination of matrix effects; only less than half of its MS signal could be recovered. The late-eluting compounds were less affected by matrix components in all conditions tested. Further, the robustness of the applied LC-MS method was checked by means of a fractional factorial design. The results indicate that small changes to the sample preparation parameters, namely pH and concentration of extraction buffer, shaking time, drying temperature and extraction volumes, did not significantly (α = 0.05) affect the recoveries of ergot alkaloids.

Fluorescent Alloyed CdZnSeS/ZnS Nanosensor for Doxorubicin Detection.

Doxorubicin (DOX) is widely used in chemotherapy as an anti-tumor drug. However, DOX is highly cardio-, neuro- and cytotoxic. For this reason, the continuous monitoring of DOX concentrations in biofluids and tissues is important. Most methods for the determination of DOX concentrations are complex and costly, and are designed to determine pure DOX. The purpose of this work is to demonstrate the capabilities of analytical nanosensors based on the quenching of the fluorescence of alloyed CdZnSeS/ZnS quantum dots (QDs) for operative DOX detection. To maximize the nanosensor quenching efficiency, the spectral features of QDs and DOX were carefully studied, and the complex nature of QD fluorescence quenching in the presence of DOX was shown. Using optimized conditions, turn-off fluorescence nanosensors for direct DOX determination in undiluted human plasma were developed. A DOX concentration of 0.5 µM in plasma was reflected in a decrease in the fluorescence intensity of QDs, stabilized with thioglycolic and 3-mercaptopropionic acids, for 5.8 and 4.4 %, respectively. The calculated Limit of Detection values were 0.08 and 0.03 µg/mL using QDs, stabilized with thioglycolic and 3-mercaptopropionic acids, respectively.

SERS Assays Based on Electrospun Nanofibers: Preparation and Analytical Applications.

Surface-enhanced Raman spectroscopy (SERS) is a powerful tool and an up-to-date method of analytical chemistry due to its high sensitivity and fingerprint recognition capabilities. Nowadays SERS due to its label-free detection capabilities is being actively developed in medical fields, for example in the analysis of biologically important substances in different matrixes, for potential on-site detection of toxic substances, food safety, and so on. To get the SERS signal, it is necessary the presence of plasmonic nanostructures in the SERS substrates. Electrospun nanofibers have been an attractive alternative to SERS-platforms due to the diversity of advantages, including ease of preparation, structure flexibility, and others. In this review, we summarized the methods of plasmonic nanostructures incorporating substrate based on electrospun nanofibers. Also, the analytical application of SERS-active electrospun nanofibers with embedded nanostructures focused on biologically significant molecules is observed in detail. Finally, the future outlook in the application of these substrates in bioanalysis as the most promising area in analytical chemistry is presented.