Applications In Analytical Chemistry Research Articles

Microdroplet Actuation via Light Line Optoelectrowetting (LL-OEW).

Meanwhile, electrowetting-on-dielectric (EWOD) is a well-known phenomenon, even often exploited in active micro-optics to change the curvature of microdroplet lenses or in analytical chemistry with digital microfluidics (DMF, lab on a chip 2.0) to move/actuate microdroplets. Optoelectrowetting (OEW) can bring more flexibility to DMF because in OEW, the operating point of the lab chip is locally controlled by a beam of light, usually impinging onto the chip perpendicularly. As opposed to pure EWOD, for OEW, none of the electrodes has to be structured, which makes the chip design and production technology simpler; the path of any actuated droplet is determined by the movement of the light spot. However, for applications in analytical chemistry, it would be helpful if the space both below as well as that above the lab chip were not obstructed by any optical components and light sources. Here, we report on the possibility to actuate droplets by laser light beams, which traverse the setup parallel to the chip surface and inside the OEW layer sequence. Since microdroplets are grabbed by this surface-parallel, nondiverging, and nonexpanded light beam, we call this principle “light line OEW” (LL-OEW).

Sugar Pills? Investigating Humphreys’ Homeopathic Specifics: Utilizing Liquid Chromatography-Tandem Mass Spectrometry for an In-Depth Look at Nineteenth-Century American Homeopathy

Frederick K. Humphreys (1816-1900), founder of Humphreys Medicine Company, appeared at the rise of both greater acceptance and popularity of the homeopathy in the United States, as opposed to more orthodox medicine. Although bolstered by the relatively high cost and low success rate of traditional medicines, homeopathic remedies soon became a source of controversy due to the largely unknown ingredient lists and contradictory claims of effectiveness. Tandem mass spectrometry analysis (MS/MS) was used to pinpoint the ingredients of four “specifics” in Humphreys’ Homeopathic Remedies. Generated spectra showed similar base peak ions for each pill, leading to the hypothesis that each remedy contained the same basic ingredients. Further MS/MS analyses identified these base peak ions as adducts of sucrose (m/z 365, 381), its related ions (m/z 433, 203, 185), and apigenin (m/z 271). When placed into a historical context, the application of chemical analyses to study these early remedies can provide insight into the question of validity that evoked arguments during the late nineteenth century.

Fluid control with hydrophobic pillars in paper-based microfluidics

Paper-based microfluidics has been widely used in chemical and medical analysis applications. In the conventional paper-based microfluidic approach, fluid is propagating inside the porous structure, and the flow direction of the fluid propagation is usually controlled with the pre-defined hydrophobic barrier (e.g. wax). However, the fluid propagation velocity inside the paper-based microfluidic devices largely depends on the material properties of paper and fluid, the relative control method is rarely reported. In this study, a fluid propagation velocity control method is proposed for paper-based microfluidics: hydrophobic pillar arrays with different configurations were deposited in the microchannels in paper-based microfluidics for flow speed control, the result indicates the deposited hydrophobic pillar arrays can effectively slow down the fluid propagation at different levels and can be used to passively control the fluid propagation inside microchannels for paper-based microfluidics. For the demonstration of the proposed fluid control methods, a paper-based microfluidic device for nitrite test in water was also fabricated. The proposed fluid control method for paper-based microfluidics may have significant importance for applications that involve sequenced reactions and more actuate fluid manipulation.

Synthesis, Evaluation, and Characterization of an Ergotamine Imprinted Styrene-Based Polymer for Potential Use as an Ergot Alkaloid Selective Adsorbent.

Alkaloid toxicities negatively impact livestock health and production. To assess alkaloid occurrences, adsorbent technologies may offer effective means to their extraction and isolation from a complex feed matrix. In this study, molecularly imprinted polymers (MIPs) were synthesized and evaluated for their specificity of binding to various ergot alkaloids. Co-polymers of styrene and hydroxyethyl methacrylate were synthesized in the absence or presence of an ergotamine (ETA) template, yielding non-imprinted polymer (NIP) and molecularly imprinted polymer (MIP), respectively. The influence of parameters such as pH, temperature, and initial concentration on the adsorption of ergot alkaloids was evaluated along with their application as solid phase extraction materials. Chemical and morphological properties were characterized. Adsorption was generally greater for MIP compared to NIP. Cross-reactivity with related alkaloids existed due to similarities in structure and functional groups and was dependent on the type and concentration of alkaloid and polymer type (alkaloid type × concentration × product; P < 0.05). The pH of the medium had no influence on the binding properties of polymers toward ETA within a pH range of 2–10. Binding was independent of temperature between 36 and 42 °C. When kinetics of adsorption were evaluated, the Langmuir isotherm had a better fit (R2 > 0.95) to adsorption equilibrium data than the Freundlich equation. The maximum amounts adsorbed (Qo) from the Langmuir model were 8.68 and 7.55 μM/g for MIP and NIP, respectively. Fourier transform infrared, scanning and tandem electron microscopy, and Brunauer–Emmett–Teller analysis confirmed a highly porous MIP structure with a greater surface area compared to NIP. Binding characteristics evaluated with computational strategy using molecular docking experiments and in vitro in a complex media (rumen fluid) indicated a stronger ETA adsorption by the tested composition selected among other polymeric materials and affinity of MIP compared with NIP. This study suggested the possible utility of MIP as a solid phase extraction sorbent for applications in analytical chemistry or sensing devices tailored to track ergot alkaloid incidence and the fate of those alkaloids in complex ruminal digestive samples.

Ionic Liquids in Analytical Chemistry: Applications and Recent Trends

Background: Ionic liquids (ILs) are a unique class of compounds consisting exclusively of cations and anions that possess distinctive properties such as low volatility, high thermal stability, miscibility with water and organic solvents, electrolytic conductivity and non-flammability. Ionic liquids have been defined as "design solvents", because it is possible to modify their physical and chemical properties by appropriately choosing cations and anions, in order to meet the specific characteristics based on their potential application. Introduction: Due of their tunable nature and properties, ILs are considered as the perfect candidates for numerous applications in analytical chemistry including sample preparation, stationary phases in liquid or gas chromatography, additives in capillary electrophoresis, or in mass spectrometry for spectral and electrochemical analysis. In the last years, the number of publications regarding ILs has rapidly increased, highlighting the broad applications of these compounds in various fields of analytical chemistry. Results: This review first described the main physico-chemical characteristics of ionic liquids, and subsequently reported the various applications in different subdisciplines of analytical chemistry, including the extraction procedure and separation techniques. Furthermore, in each paragraph the most recent applications of ionic liquids in the food, environmental, biological, etc. fields have been described. Conclusion: Overall, the topic discussed highlights the key role of ionic liquids in analytical chemistry, giving hints for their future applications in chemistry but also in biology and medicine.

A new grey relational analysis application in analytical chemistry: Natural deep eutectic solvent as a green extractant for HPLC determination of lamotrigine in plasma

Developing new, eco-friendly solvents which would meet technological and economic demands is perhaps the most popular aspects of Green Chemistry. Natural deep eutectic solvents (NADES) fully represent green chemistry principles. In this sense a new green vortex-assisted NADES-based microextraction (VA-NADES-ME) was established, for the first time, for the extraction of lamotrigine from human plasma samples using NADES, as a green extractant alternative to hazard organic solvents. Then extract was determined by high performance liquid chromatography with ultraviolet detection (HPLC–UV). The mixture of (choline chloride/ethylene glycol) as NADES was found to be the best candidate for extracting lamotrigine. Grey relational analysis-based Taguchi method was utilized as a chemometric protocol for multiobjective optimization of different HPLC performance parameters. The optimum mobile phase was a mixture of water with 0.3 % triethylamine in 0.02 M H3PO4 (adjusted to pH 5.5) and acetonitrile (70:30 v/v) with an overall pH 6. Finally, the confirmation test was performed to validate the predicted optimal results and established an improvement of 32.84 % from the initial design. The mobile phase was eluted in an isocratic mode with a flow rate of 0.8 mL/min and UV detection at 310 nm. The retention times of lamotrigine and tinidazole (internal standard) were 4.7 and 3.4 min, respectively. Proposed method was linear over the concentration range of 0.1–15.0 μg mL−1. The greenness profile was assessed using Analytical GREEnness metric approach which assured the greenness of the proposed method compared with other reported methods.

SURFACE-ENHANCED RAMAN SCATTERING ACTIVITY OF PLASMONIC Ag–Ti NANOISLAND FILMS

Bimetallic nanostructure of noble metals is an alternative material that can provide the tenability of plasmonic performance. In this study, the plasmonic silver–titanium nanoisland (Ag–Ti NI) films deposited on a silicon wafer and glass slide substrates were prepared by magnetron co-sputtering of high-purity Ag target (99.99%) operated at 100 W-DC source, and Ti target (99.99%) operated at 50–250 W-DC pulsed source. The surface morphologies of the prepared films revealed a noncontinuous island Ag–Ti according to the formation of thin film growth based on the Volmer–Weber model. An increase in the sputtering power of the Ti target caused an evident increase in the Ag–Ti NIs diameter. The localized surface plasmon resonance (LSPR) was evaluated by UV–Vis–NIR spectrophotometry. The LSPR peak shift disappears with an increase of the sputtering power of the Ti target.In addition, the results confirmed that the surface-enhanced Raman scattering (SERS) activity of the bimetallic Ag–Ti NIs significantly improved in performance and stability, which is promising for the application in analytical chemistry.

Quo vadis Spectroscopy?

Since the early days of modern chemistry, analytical chemistry always tried to offer solutions to real-life problems. This is how, in the midst of the Industrial Revolution, Margueritte developed the titrimetric determination of iron using the chameleon solution (potassium permanganate). In 1860, for the first time, Bunsen and Kirchhoff used element-specific light emission and absorption of flame-evaporated alkali metal salts for qualitative analysis. Their discovery was groundbreaking because the analytical sensitivity and detection capability of the proposed method was several orders of magnitude lower than that of the contemporary classical analytical ones. Application of this method, leading to the establishment of spectroscopic techniques, also allowed the discovery of about ten chemical elements. Another timeless merit of spectroscopy is the ability to determine the elemental composition of a star in a distant galaxy. In the second half of the 20th century, an era of rapid development of instrumental analysis, atomic spectroscopy also brought several revolutionary results. One such breakthrough was the reduction of the sample volume required for analysis down to microliters with the introduction of graphite furnace atomic absorption spectrometry (GFAAS). The detection limit of GFAAS also decreased by several orders of magnitude. Another important advance was the combination of inductively coupled plasma as a high-temperature ion source with a mass spectrometer (ICP-MS). In the late 1980s, the hyphenation of atomic spectrometric devices to chromatographs, aiming at elemental speciation, also emerged. In Central and Eastern Europe, spectroscopic research was driven by the embargo of the Coordinating Committee for Multilateral Export Controls established by the Western Bloc during the Cold War. In this emerging era, scientific research and applications went hand in hand. Therefore, the science education program and fundamental research were very strong in those countries, contributing considerably to the development of spectrochemistry in Hungary. By the end of the 20th century, development of new equipment and procedures was increasingly carried out by instrument manufacturers, and fundamental research at universities and institutes was relegated to second place. Analytical chemistry has experienced a considerable shift from determination of inorganic compounds and small organic molecules towards that of large (bio)molecules. Expansion of the application of analytical chemistry to forensic, environmental and biochemical questions has been observed and the role of atomic spectrometric techniques seemed to fade away. However, sheer use of these high-performance instruments is not always appropriate or cost-effective; the reliability of the results and elimination of interference must be thoroughly explored. However, when publishing such results, there is a risk that our communications will be rejected due to lack of novelty. Nevertheless, carrying out fundamental research cannot be avoided, as it is not possible to offer appropriate decisions to stakeholders based on questionable results. Presently, GFAAS and ICP-MS allow reliable quantitative determination of virtually any element in any sample. Recently, microwave plasma using nitrogen isolated from the air was launched on the market. This device requires significantly lower operating costs compared to the conventional ICP and reliable simultaneous multi-element analysis has also become possible. Another promising direction is single-particle ICP-MS applied for the characterization of inorganic nanoparticles. Most of the articles of the current issue of BrJAC also demonstrate that atomic spectrometry has become indispensable in many areas of our life. Enjoy reading the current issue!

Upscaling production of droplets and magnetic particles with additive manufacturing

PurposeMonodisperse microfluidic emulsions – droplets in another immiscible liquid – are beneficial to various technological applications in analytical chemistry, material and chemical engineering, biology and medicine. Upscaling the mass production of micron-sized monodisperse emulsions, however, has been a challenge because of the complexity and technical difficulty of fabricating or upscaling three-dimensional (3 D) microfluidic structures on a chip. Therefore, the authors develop a fluid dynamical design that uses a standard and straightforward 3 D printer for the mass production of monodisperse droplets.Design/methodology/approachThe authors combine additive manufacturing, fluid dynamical design and suitable surface treatment to create an easy-to-fabricate device for the upscaling production of monodisperse emulsions. Considering hydrodynamic networks and associated flow resistance, the authors adapt microfluidic flow-focusing junctions to produce (water-in-oil) emulsions in parallel in one integrated fluidic device, under suitable flow rates and channel sizes.FindingsThe device consists of 32 droplet-makers in parallel and is capable of mass-producing 14 L/day of monodisperse emulsions. This convenient method can produce 50,000 millimetric droplets per hour. Finally, the authors extend the current 3 D printed fluidics with the generated emulsions to synthesize magnetic microspheres.Originality/valueCombining additive manufacturing and hydrodynamical concepts and designs, the authors experimentally demonstrate a facile method of upscaling the production of useful monodisperse emulsions. The design and approach will be beneficial for mass productions of smart and functional microfluidic materials useful in a myriad of applications.

Flow-based methods and their applications in chemical analysis

Flow-based methods and their applications in chemical analysis

Development of microfluidic droplet shooter and its application to interface for mass spectrometry

Gas/liquid microfluidics has developed for high-efficiency mass transport, mixing and reaction in various applications of chemical analysis and synthesis. However, ejection of uniform liquid droplets floating in gas phase from the microchannels with trajectory control is still challenging. In this study, we developed a method for shooting picoliter droplets in a controlled direction that utilizes microscale gas-phase laminar flows and applied it to a mass spectrometry (MS) interface. The principle of droplet shooting using two-step gas-phase flow focusing was proposed. A microfluidic device with a branched and stepped hydrophobic microchannel was fabricated by top-down glass fabrication technologies. We verified the shooting of droplets of aqueous and organic solvents in the volume range of 3.61–24.9 pL at kilohertz frequencies. A model that considers the capillary force and the drag force was verified to establish a design guideline. Utilizing the developed microfluidic droplet shooter, we demonstrated high-efficiency sample transport to a single quadrupole mass spectrometer. We verified a sample injection rate of approximately 100 % by ejection of picoliter droplets into an MS injection aperture of a 400-μm diameter and achieved detection of caffeine with 3 times higher sensitivity than conventional electrospray ionization interface with sample dispersion.

Self-Powered Respiration Monitoring Enabled By a Triboelectric Nanogenerator.

In mammals, physiological respiration involves respiratory cycles of inhaled and exhaled breaths, which has traditionally been an underutilized resource potentially encompassing a wealth of physiologically relevant information as well as clues to potential diseases. Recently, triboelectric nanogenerators (TENGs) have been widely adopted for self-powered respiration monitoring owing to their compelling features, such as decent biocompatibility, wearing comfort, low-cost, and high sensitivity to respiration activities in the aspect of low frequency and slight amplitude body motions. Physiological respiration behaviors and exhaled chemical regents can be precisely and continuously monitored by TENG-based respiration sensors for personalized health care. This article presents an overview of TENG enabled self-powered respiration monitoring, with a focus on the working principle, sensing materials, functional structures, and related applications in both physical respiration motion detection and chemical breath analysis. Concepts and approaches for acquisition of physical information associated with respiratory rate and depth are covered in the first part. Then the sensing mechanism, theoretical modeling, and applications related to detection of chemicals released from breathing gases are systemically summarized. Finally, the opportunities and challenges of triboelectric effect enabled self-powered respiration monitoring are comprehensively discussed and criticized.

The Dilution Effect in High-Power Disk Laser Welding the Steel Plate Using a Nickel-Based Filler Wire

High-power disk laser welding a steel plate using the Inconel 82 (IN82) filler wire with a 1.2 and 3.0 m/min feeding rate has been investigated in the experiment. The rapid thermal cycle combined with convection induced by the keyhole mechanism in laser welding results in the rapid solidification of the fusion zone (FZ). However, the microstructure of the FZ is not homogeneous at the macroscopic scale. The dilution of the FZ is important in determining the final microstructure of the weldment. For the specimen with a 1.2 m/min wire feeding rate, a lower amount of Ni-based IN82 filler is introduced into the weld pool, and the dilution of the FZ is between 65% and 100%. The BCC structure with high density boundaries dominates the entire FZ. For the specimen with a 3.0 m/min wire feeding rate, part of the filler melt is trapped on the top of the weld pool, and solidified into austenite alloyed with a Ni concentration above 15 at%. The range of dilution in the FZ with a 3.0 m/min wire feeding rate is decreased to 50–90%. There are hot cracks initiated/propagated along interdendritic austenite and in the austenite free of boundaries. Boundaries, especially for high-angle ones, in the BCC structure retard hot crack propagation in the FZ. The application of quantitative chemical analyses of Fe or Ni concentrations in the weldment provides a good approximation in evaluating the dilution of the FZ in laser welding. The methodology proposed in this study shows potential to obtain the dilution of any specific location in the FZ for industrial application in the future.

Ceria Nanozyme and Phosphate Prodrugs: Drug Synthesis through Enzyme Mimicry.

Nanozymes can mimic the activities of diverse enzymes, and this ability finds applications in analytical sciences and industrial chemistry, as well as in biomedical applications. Among the latter, prodrug conversion mediated by nanozymes is investigated as a step toward site-specific drug synthesis, to achieve localized therapeutic effects. In this work, we investigated a ceria nanozyme as a mimic to phosphatase, to mediate conversion of phosphate prodrugs into corresponding therapeutics. To this end, the substrate scope of ceria as a phosphatase mimic was analyzed using a broad range of natural phosphor(di)esters and pyrophosphates. Knowledge of this scope guided the selection of existing phosphate prodrugs that can be converted by ceria into the corresponding therapeutics. "Extended scaffold phosphates" were engineered using self-immolative linkers to accommodate a prodrug design for amine-containing drugs, such as monomethyl auristatin E. Phosphate prodrugs masked activity of the toxin, whereas prodrug conversion mediated by the nanozyme restored drug toxicity, which was validated in mammalian cell culture. The main novelty of this work lies in the rational pairing of the ceria nanozyme with the existing and the de novo designed "extended scaffold" phosphate prodrugs toward their use in nanozyme-prodrug therapy based on the defined nanozyme substrate scope.

Integrating the MasSpec Pen with Sub-Atmospheric Pressure Chemical Ionization for Rapid Chemical Analysis and Forensic Applications.

Analytical methods that allow rapid, sensitive, and specific chemical measurements are central to forensic analysis and essential to accelerating compound screening and confirmation. We have previously reported the development of the MasSpec Pen technology as an easy-to-use and disposable hand-held device integrated to a mass spectrometer for direct analysis and molecular profiling of biological samples. In this Technical Note, we describe a new apparatus that integrates the MasSpec Pen device with a subatmospheric pressure chemical ionization (sub-APCI) source and an ion trap mass spectrometer for detection and semiquantitative analysis of forensic-related compounds. Coupling the MasSpec Pen device to a sub-APCI source allowed semiquantitative analysis of the drugs cocaine and oxycodone, the agrochemicals atrazine and azoxystrobin, and the explosives trinitrotoluene and dinitroglycerin in under 20 s. Using chemical ionization, improved reproducibility and sensitivity for targeted chemical detection and compound identification was achieved while maintaining the user-friendly features of the hand-held MasSpec Pen device. Limits of detection in the high picogram to low nanogram range were obtained for the compounds analyzed, which are within the range of federal screening cutoffs and those reported for other ambient ionization MS techniques. Altogether, the MasSpec Pen sub-APCI system described enabled rapid and semiquantitative chemical analysis for forensic applications and could be further adapted and applied to other areas of chemical testing.

Preparation of Noble Metal Nanoparticles and Hydrogel Composite Materials and Their Application in Analytical Chemistry

Abstract Noble metal nanomaterials are widely used in analytical chemistry due to their unique physical and chemical properties. However, the instability of nanoparticles in aqueous solutions limits the development of noble metal nanomaterials. Hydrogels acting as nanoparticle carriers can solve this problem well. Due to the excellent mechanical properties, good biocompatibility and ease of modification, hydrogels can improve the properties of nanomaterials and provide new application advantages. Therefore, more and more researches focus on the development of noble metal nanoparticles and hydrogel composite materials. Here, we reviewed the preparation principles and application progress of various types of noble metal nanoparticles and hydrogel composite materials in the past five years, and discussed the application advantages of composite materials and their further research orientations.

Analytical Chemistry and Innovative Applications

In this issue of the Journal “Analytica”, there is a selection of accepted articles, after peer-review process, in which the great importance that Analytical Chemistry plays in the field of Applied Sciences and the vastness of the implications in the various sectors are highlighted [...]

Nanomaterial-enhanced 3D-printed sensor platform for simultaneous detection of atrazine and acetochlor

The widespread use of herbicides in agriculture and gardening causes environmental and safety issues such as water pollution. Thus, efficient and convenient analysis of the levels of herbicide residues is of significant importance. Here, we employed 3D-printing to design a multiplex immunosensor for simultaneous detection of two widely used herbicides, atrazine and acetochlor. Multiplexing was achieved through customization of a lateral flow immunoassay, and then integrated with an electrochemical analyzer for ultrasensitive detection. Quantification of herbicide residues was realized through the detection of a novel nanomaterial label, the mesoporous core-shell palladium@platium nanoparticle (Pd@Pt NP), for its outstanding peroxidase-like property. During the electrochemical analysis, the catalytic activity of Pd@Pt NPs on the redox reaction between thionin acetate and hydrogen peroxide provided an electrochemically driven signal that accurately indicated the level of herbicide residues. Using this Nanomaterial-enhanced multiplex electrochemical immunosensing (NEMEIS) system, simultaneous detection of atrazine and acetochlor was realized with a limit of detection of 0.24 ppb and 3.2 ppb, respectively. To further evaluate the feasibility, the optimized NEMEIS was employed for detection in atrazine and acetochlor residue-containing spiked samples, and an overall recovery with 90.8% – 117% range was obtained. The NEMEIS constructed with the aid of 3D-printing provides a rapid, precise, economical, and portable detection device for herbicides, and its success suggests potential broad applications in chemical analysis, biosensors and point-of-care monitoring.

Generalized Read-Across prediction using genra-py.

Generalized Read-Across (GenRA) is a data-driven approach to estimate physico-chemical, biological or eco-toxicological properties of chemicals by inference from analogues. GenRA attempts to mimic a human expert's manual read-across reasoning for filling data gaps about new chemicals from known chemicals with an interpretable and automated approach based on nearest-neighbors. A key objective of GenRA is to systematically explore different choices of input data selection and neighborhood definition to objectively evaluate predictive performance of automated read-across estimates of chemical properties. We have implemented genra-py as a python package that can be freely used for chemical safety analysis and risk assessment applications. Automated read-across prediction in genra-py conforms to the scikit-learn machine learning library's estimator design pattern, making it easy to use and integrate in computational pipelines. We demonstrate the data-driven application of genra-py to address two key human health risk assessment problems namely: hazard identification and point of departure estimation. The package is available from github.com/i-shah/genra-py.

Facile and highly selective sensing of hypochlorous acid in aqueous solution and living cells by using as-prepared WSe2 quantum dots

Abstract The monitoring of biological hypochlorous acid (HClO) in living cells is vital in clarifying its physiological functions. Generally, HClO fluorescent probes that can be used for intracellular detection are small organic molecules that require complex synthesis steps. Herein, the as-prepared WSe2 quantum dots (QDs) without any further modification was verified to be a HClO fluorescent probe due to the unique chemical property of selenium. Compared with molecular probes, WSe2 QDs displayed improved performance in HClO detection, such as the low limit of detection (1.1 nM), high selectivity, fast response, good aqueous solubility, friendly operation, excellent photostability, and pH-independent response. Furthermore, the most outstanding advantage of as-prepared WSe2 QDs was their ready availability with up to the gram-large scale. WSe2 QDs were successfully used for the detection of exogenous and endogenous HClO in living cells. Meanwhile, this probe was applied to discriminate normal cells and cancerous cells on the basal of their different HClO levels. The sensing mechanism of WSe2 QDs to HClO was further carefully demonstrated and proposed to be HClO induced the oxidation and degradation of WSe2 QDs into non-fluorescent products. We anticipate that the as-prepared WSe2 QDs should have broad applications in chemical analysis and revealing the physiological function of HClO in vivo.