Paper-based colorimetric sensor using Photoshop and a smartphone app for the quantitative detection of carbofuran.

We developed a smartphone-assisted microchemistry analyzer for the quantitative detection of carbofuran using a paper-based colorimetric sensor, Photoshop software, and a smartphone app. The changes in color of the carbofuran enzymatic reaction in the paper-based sensor were captured and analyzed using a smartphone-controlled analyzer with an LED light source and a smartphone camera. The high accuracy of this method was demonstrated for the determination of carbofuran with a linear response in the range 0.05-1.0 ppm and limits of detection (LOD) of 0.02 and 0.018 ppm using Photoshop and smartphone app colorimetric analysis, respectively. These two methods not only show the high sensitivity and highly quantitative relationships between the concentrations of commercial carbofuran and characteristic color values of the blue channel in smartphone images but were also applied to infusions of tea. Moreover, the smartphone app is able to GPS tag the location of the test and transmit the results to a website that displays quantitative results from carbofuran samples on a map.

Heterogeneity of otolith chemical composition from two-dimensional mapping: Relationship with biomineralization mechanisms and implications for microchemistry analyses.

Although otoliths are widely used as archives to infer life-history traits and habitat use in fishes, their biomineralization process remains poorly understood. This lack of knowledge is problematic as it can lead to misinterpretation of the different types of signals (e.g., optical or chemical) that provide basic data for research in fish ecology, fisheries management, and species conservation. Otolith calcification relies on a complex system involving a pericrystalline fluid, the endolymph, whose organic and inorganic compositions are spatially heterogeneous for some constituents. This property stems from the particular structure of the calcifying saccular epithelium. In this study, we explored the spatial heterogeneity of elemental incorporation in otoliths for two species of high economic interest, European hake Merluccius merluccius (L. 1758) and European sea bass Dicentrarchus labrax (L. 1758). Two-dimensional mappings of chemical elements were obtained using UV high-repetition-rate femtosecond laser ablation (fs-LA) system coupled to a high-resolution inductively coupled plasma sector field mass spectrometer analyses on transverse sections of sagittae. Results highlighted a clear asymmetry between proximal (sulcus) and distal (antisulcus) concentrations for elements such as magnesium (Mg), phosphorus (P), manganese (Mn), and potassium (K) with concentration gradient directions that varied depending on the element. Strontium (Sr) and barium (Ba) did not show a proximo-distal gradient. These results are discussed in light of current knowledge on the endolymph composition and the mechanisms that lead to its compartmentalization, highlighting the need for further research on otolith biomineralization. Operational implications for studies based on otolith chemical composition are also discussed with emphasis on advice for sampling strategies to avoid analytical biases and the need for in-depth analyses of analytical settings before comparing otolith signatures between species or geographical areas.

Otolith microchemistry reveals diverse habitat uses and migratory patterns of two Coilia species (Engraulidae) in the Min River Estuary, southern China.

A recent study based on gonad histology revealed that the existence of the spawning grounds for Gray's grenadier anchovy (Coilia grayii) and Osbeck's grenadier anchovy (C. mystus) in the Min River Estuary, the largest in Fujian Province, southern China. Further confirming their natal sources and migratory patterns is essential to understand their life histories. We used otolith microchemistry to assess the origins and habitat uses of 23 C. grayii and 22 C. mystus, collected the Min River Estuary and the adjacent waters. The results showed that C. grayii spawned in both freshwater (n| = 14) and brackish water (n = 9), and C. mystus spawned mainly in brackish water (n| = 20) with minor in freshwater (n| = 1) and marine water (n| = 1). The migratory patterns of C. grayii (four types) and C. mystus (five types) were diverse, mainly exhibiting anadromous and semi-anadromous behaviors. The first migratory behavior of C. grayii and C. mystus occurred within the age of the first year. The findings have significant implications for fishery stock management of the Min River Estuary and its adjacent waters.

Otolith microchemistry of freshwater indigenous minor carp (Bangana dero) as a biomonitoring tool to trace heavy metals in River Sutlej, Ropar Wetland (Ramsar site), Punjab, India.

The elemental composition of the fish otolith may represent a permanent record of the environmental condition the fish inhabited. Fish otolith grows incrementally from the core to a marginal region that acts as a repository of trace metal signatures. The present study explores the potential application of otolith microchemistry of the benthopelagic indigenous minor carp Bangana dero sampled from the Ropar wetland on River Sutlej, Punjab. The concentration of sixteen metals was evaluated in the otolith (n = 42) and water (n = 48) for the post-monsoon and pre-monsoon season from 2020 to 2022 using inductively coupled plasma mass spectrometry (ICP-MS) followed by element detection in the core and marginal region of whole otolith, using energy-dispersive mass spectroscopy (EDS). All the heavy metals exhibited an increase in metal concentrations in fish otolith than water during the post-monsoon season. By indices approach, the otolith was found to have a high bioaccumulation factor for Se in the post-monsoon and Hg in the pre-monsoon. Certain trace metals like As and Hg exhibited fluctuations in their core and marginal region. Thus, trace metal patterns in the otolith could act as a potential tool for monitoring the seasonal changes of metals in water bodies. The EFHg, EFSe and EFAs in the fish otolith predicted its anthropogenic source, while the remaining studied elements showed ambient water origin. Thus, using the otoliths of Bangana dero as a long-term monitoring tool in the future may be helpful for environmental assessments and the reconstruction of historical exposure for safeguarding of water bodies.

A microscale soft ionic power source modulates neuronal network activity.

Bio-integrated devices need power sources to operate1,2. Despite widely used technologies that can provide power to large-scale targets, such as wired energy supplies from batteries or wireless energy transduction3, a need to efficiently stimulate cells and tissues on the microscale is still pressing. The ideal miniaturized power source should be biocompatible, mechanically flexible and able to generate an ionic current for biological stimulation, instead of using electron flow as in conventional electronic devices4-6. One approach is to use soft power sources inspired by the electrical eel7,8; however, power sources that combine the required capabilities have not yet been produced, because it is challenging to obtain miniaturized units that both conserve contained energy before usage and are easily triggered to produce an energy output. Here we develop a miniaturized soft power source by depositing lipid-supported networks of nanolitre hydrogel droplets that use internal ion gradients to generate energy. Compared to the original eel-inspired design7, our approach can shrink the volume of a power unit by more than 105-fold and it can store energy for longer than 24 h, enabling operation on-demand with a 680-fold greater power density of about 1,300 W m-3. Our droplet device can serve as a biocompatible and biological ionic current source to modulate neuronal network activity in three-dimensional neural microtissues and in ex vivo mouse brain slices. Ultimately, our soft microscale ionotronic device might be integrated into living organisms.

Effect of metabolic rate on time-lag changes in otolith microchemistry: an experimental approach using Salmo trutta.

Ecologists have long been interested in relevant techniques to track the field movement patterns of fish. The elemental composition of otoliths represents a permanent record of the growing habitats experienced by a fish throughout its lifetime and is increasingly used in the literature. The lack of a predictive and mechanistic understanding of the individual kinematics underlying ion incorporation/depletion limits our fine-scale temporal interpretation of the chemical signal recorded in the otolith. In particular, the rate at which elements are incorporated into otoliths is hypothesized to depend on fish physiology. However, to date, time lags have mostly been quantified on a population scale. Here, we report results from controlled experiments (translocation and artificially enriched environment) on individual trace element incorporation/depletion rates in Salmo trutta (Salmonidae). We reported significant lags (i.e. weeks to months) between changes in water chemistry and the subsequent change in otolith composition and highlighted substantial inter-individual variations in the timing and magnitude of Sr/Ca and Ba/Ca responses. These differences are partially linked to the energetic status (i.e. metabolic rate) of the individuals. It therefore appears that individuals with the highest metabolic rate are more likely to record detailed (i.e. brief) temporal changes than individuals having lower metabolic values. The time taken for environmental changes to be reflected in the growing otolith thus can no longer be assumed to remain a constant within populations. Results from the current study are a step towards the fine reconstruction of environmental histories in dynamic environments.

3D (x-y-t) Raman imaging of tomato fruit cuticle: Microchemistry during development.

The cuticle is a protective extracellular matrix that covers the above-ground epidermis of land plants. Here, we studied the cuticle of tomato (Solanum lycopersicum L.) fruits in situ using confocal Raman microscopy. Microsections from cuticles isolated at different developmental stages were scanned to visualize cuticle components with a spatial resolution of 342 nm by univariate and multivariate data analysis. Three main components, cutin, polysaccharides, and aromatics, were identified, with the latter exhibiting the strongest Raman scattering intensity. Phenolic acids and flavonoids were differentiated within the cuticle, and three schematic cuticle models were identified during development. Phenolic acids were found across the entire cuticle at the earliest stage of development, i.e. during the formation of the procuticle layer. Based on a mixture analysis with reference component spectra, the phenolic acids were identified as mainly esterified p-coumaric acid together with free p-hydroxybenzoic acid. During the cell expansion period of growth, phenolic acids accumulated in an outermost layer of the cuticle and in the middle region of the pegs. In these stages of development, cellulose and pectin were detected next to the inner cuticle region, close to the epidermal cell where flavonoid impregnation started during ripening. In the first ripening stage, chalconaringenin was observed, while methoxylated chalcones were chosen by the algorithm to fit the mature cuticle spectra. The colocation of carbohydrates, esterified p-coumaric acid, and methoxylated chalconaringenin suggests that the latter two link polysaccharide and cutin domains. Elucidating the different distribution of aromatics within the cuticle, suggests important functions: (1) overall impregnation conferring mechanical and thermal functions (2) the outermost phenolic acid layer displaying UV-B protection of the plant tissue.

European flounder foraging movements in an estuarine nursery seascape inferred from otolith microchemistry and stable isotopes.

Despite the importance of estuarine nurseries in the regulation of many fish stocks, temporal and spatial movements and habitat use patterns of juvenile fish remain poorly understood. Overall, combining several movement metrics allowed us to characterize dispersal patterns of juvenile flounder, Platichthys flesus, along an estuarine seascape. Specifically, we investigated otolith microchemistry signatures (Sr:Ca and Ba:Ca ratios) and stable isotope ratios (δ13C and δ15N) in muscles of these juveniles, during three consecutive years to assess inter-annual fluctuations in their home range and isotopic niches. The morphological condition and lipid content of individuals were lower in years of high as compared to low dispersal along the estuarine gradient. We discuss these results in relation to the ecosystem productivity and intra- and inter-specific competition level, which in turn affects movements and foraging behaviors of juvenile flounders.

Multivariate analysis of otolith microchemistry can discriminate the source of oil contamination in exposed fish.

The uptake of metals into the aragonite lattice of the fish otolith (ear-bone) has been used for decades as a historical record of exposure to metals in polluted environments. The relative abundance of two metals in particular, Ni and V, are used in forensic chemical analysis of crude oils to assist in confirming its origin. In this study we investigate the potential for metal accumulation in otoliths to act as a biomarker of exposure to crude oil. Using a 33-day static-renewal laboratory trial design, 56 juvenile Lates calcarifer (commonly known as Asian seabass or barramundi) were fed diets enriched with V (20 mg/kg), Ni (500 mg/kg), Fe (500 mg/kg), and two crude oils with distinctly different metals profiles: a heavy fuel oil (1% w/w) and a typical Australian medium crude (1% w/w). Fish exposed to crude oils showed Ba and Al retained in otoliths in a dose-dependent manner, but fish fed V-, Ni- and Fe-enriched diets showed no metal increase in otoliths, indicating that V, Ni and Fe are not incorporated into the otolith of L. calcarifer via dietary exposure. For crude oils, incorporation into otolith for many metals is likely limited due to porphyrin casing reducing their bioavailability. Principal components analysis (PCA) and subsequent linear discriminatory analysis (LDA) of selected otolith metals demonstrated that, even despite large variability in the metal abundances detected in otolith between individuals within the test groups (cv = 1.00), it is possible to discriminate between fish exposed to different crude oils using multivariate analysis of their otolith microchemistry.

Fish otolith microchemistry as a biomarker of the world's largest mining disaster.

The Fundão dam collapse occurred in late 2015, resulting in the largest disaster in the world involving mine tailings, releasing at least 50 million m3 of iron ore tailings into the Doce river basin (Brazil). Studies realized along the Doce river after the disaster showed high concentrations of some elements above recommended Brazilian standards in different environmental matrices such as Al, Ba, Co, Fe, Hg, Mn, and Ni. This study aims to test the hypothesis that otolith microchemistry can be used as a pollution biomarker over time, that is, the otoliths sampled in the directly impacted area (Doce estuary) will present higher concentrations of the chemical elements in relation to the two other indirectly impacted estuaries (Ipiranga and São Mateus). The marine catfish Genidens genidens was chosen as a sentinel species, as this species presents several features that characterize it as a key species of the estuarine environment. The hypothesis was tested from a comparison between estuaries of the element/Ca ratios (Ba, Fe, and Mn) from the otolith edge (representing more recent deposition) and through the elemental composition profile, from core to edge of otolith. For the otolith edge analyses, it was possible to determine significant differences between estuaries in all three element:Ca ratios (Ba, Fe, and Mn). For the profile analysis (from core to edge), the Doce river showed consistent concentrations of Fe:Ca and Ba:Ca ratios higher than those of the Ipiranga and São Mateus estuaries throughout the life of the fish. In otoliths sampled in the Doce estuary, the possible identified peak of the Ba:Ca and Mn:Ca ratios, was probably associated with the Fundão Dam disaster. The present study showed that otolith microchemistry can be used as a sclerochronological biomarker of metallic aquatic pollution.

Habitat use and nursery evaluation for the longnose stingray Hypanus guttatus (Bloch & Schneider, 1801) using vertebral microchemistry.

Here, we use vertebral microchemistry to investigate the habitat use patterns of the longnose stingray Hypanus guttatus in the northern and northeastern regions of Brazil, and to evaluate the existence of potential nurseries of the species. Samples were collected in Maranhão, Rio Grande do Norte and Pernambuco states between 2008 and 2019. Trace element concentrations of Ba:Ca, Mg:Ca, Mn:Ca and Sr:Ca in vertebrae were determined through laser ablation inductively coupled plasma mass spectrometry. We used one-step permutational ANOVAs to evaluate multi and single-element differences in element:Ca ratios among life stages and sexes across and within sites. Element signatures differed among all sites for both single and multi-element settings. However, there were few differences among life stages within sites, except for Mn:Ca between life stages for Maranhão state, and between sexes for Sr:Ca for both Pernambuco and Rio Grande do Norte, and Ba:Ca for Rio Grande do Norte. Furthermore, all multi and single-element analyses across locations were significant and highlight the strong differences by sites. We also performed a nonmetric multidimensional scaling analysis, which demonstrated the strong differences for samples from Rio Grande do Norte and the other two sites. By analysing the lifetime transects of each sample, we observed that there was little variation in all element:Ca ratios within sites. Therefore, we argue that the longnose stingray likely does not display extensive habitat use shifts and fulfils much of its life cycle within each area. Finally, we obtained no evidence of nurseries for any site and suggest this is likely a pattern across the species distribution. Future studies investigating habitat use with samples from larger specimens (disc width > 90 cm, >15 years) are needed to better understand the species habitat use patterns across all size ranges.

Study on the pharmacokinetics of mulberry fruit polysaccharides through fluorescence labeling.

A sensitive and efficient fluorescence labeling method was developed and validated for the microanalysis and detection of polysaccharides. Fluorescein isothiocyanate (FITC) was successfully labeled on mulberry fruit polysaccharides (MFP) through a reductive amination reaction with the assistant of tyramine. The fluorescent labeled polysaccharides (FMFP) was identified by fluorescence, UV-visible, flourier transform infrared (FT-IR) and 1H NMR spectrum. Results demonstrated that the labeling efficiency of FMFP was 0.32%, and the FMFP was stable in simulated digestion fluid without cytotoxicity. The pharmacokinetics and biodistribution after administration were analyzed in rats, which indicated that the FMFP obtained could be absorbed in a short time (tmax 0.50 h) but eliminated slowly (t1/2 8.77 ± 1.38 h). At 24 h after administration, the polysaccharide could be tested mainly in intestine, stomach, liver and kidney. The FITC labeling method lays a foundation for investigating the absorption and metabolism of MFP, and provides references for the microanalysis research of bioactive polysaccharides.

Interactions between Phosphatidylcholine and Kaempferol or Myristicin: Langmuir Monolayers and Microelectrophoretic Studies.

Flavonoid compounds are known for their antibacterial, anti-inflammatory, and anticancer properties. Therefore, they can influence membrane properties that interest us, modifying both their structure and functions. We used kaempferol (K) and myricetin (M) as representatives of this group. We investigated the influence of the abovementioned compounds on model cell membranes' properties (i.e., Langmuir monolayers and liposomes). The basic research methods used in these studies were the Langmuir method with Brewster angle microscopy and microelectrophoresis. The π-A isotherms were registered for the pure components and mixtures of these compounds with phosphatidylcholine (PC) in appropriate volume ratios. Using mathematical equations, we established that kaempferol, myricetin, and the lipids formed complexes at 1:1 ratios. We derived the parameters characterizing the formed complexes, i.e., the surfaces occupied by the complexes and the stability constants of the formed complexes. Using the microelectrophoretic method, we determined the dependence of the lipid membranes' surface charge density as a function of the pH (in the range of 2 to 10) of the electrolyte solution. The presented results indicate that the PC membrane's modification with kaempferol or myricetin affected changes in the surface charge density and isoelectric point values.

An antimony-phosphomolybdate microassay of ATPase activity through the detection of inorganic phosphate.

Colorimetric methods are convenient for the determination of inorganic phosphate. However, the acidic conditions required can complicate measurement of ATPase through non-enzymatic ATP hydrolysis. Here we present an optimized antimony-phosphomolybdate microassay for the simple and rapid detection of ATPase activity, with micromolar sensitivity. The low acidity of the color reagent results in no interference for samples containing up to 0.5-5 mM ATP, dependent on the sample volume. The assay is compatible with common assay conditions and was similar in accuracy to an established continuous method. The simplicity of this method makes it ideal for medium to high throughput applications.

Microkinetic coagulation assays for human and zebrafish plasma.

Coagulation assays, prothrombin time (PT), and partial thromboplastin time (PTT) are tests to measure the clotting ability of plasma and used in evaluating patients suffering from bleeding disorders. These assays require 100 µl of human plasma. In zebrafish, dilute plasma with exogenously added human fibrinogen was used. Our objective is to create a microkinetic coagulation assay for human and zebrafish plasmas using 1 µl plasma under conditions similar to PT and PTTs. Here, we developed an assay using the Take3 plate with wells holding up to 6 µl, which can be loaded in a microplate reader for measuring the absorbance of fibrin formation. In this assay, we used 1 µl of citrated zebrafish or human plasma followed by the addition of either thromboplastin or Dade ACTIN or factor X activator from Russell viper venom as an activating agent and CaCl2. We found 4 or 3 µl of the final volume of reaction was optimal. Our results showed both zebrafish and human plasmas yielded kinetic PT, kinetic PTT, and kinetic Russel's viper venom time curves similar to previously established curves using dilute plasma. This kinetic coagulation was inhibited by heparin and was reduced significantly in coagulation factor deficient plasmas. These results validated our microkinetic coagulation assays. Moreover, we derived clotting times from these kinetic curves, which were identical to human PT, PTT, and Russel's viper venom time. In conclusion, we established a microkinetic assay that could measure blood coagulation activity in models like zebrafish and human blood samples obtained from a finger prick in adults or heel prick in infants.

High-throughput and uniform large field-of-view multichannel fluorescence microscopy with super-thin dichroism for a dPCR gene chip.

With the rapid development of digital precision medicine, the digital polymerase chain reaction (dPCR) deoxyribonucleic acid (DNA) gene chip integrates more channels with smaller size and larger area, which leads to a higher technical requirement for commercial optical fluorescence microscopy. The multitime image splicing method is widely used for DNA detection. However, it consumes time and has visible seamless image results. This work has demonstrated the design and fabrication of a three channel reversed and reduced fluorescence microscopic imaging system with high-resolution and large field of view for one-time imaging. We introduced the super ultra-thin dichroic mirror into the space between the objective lens and the gene chip to achieve a uniform illumination and a strong signal for the large area gene chip. The fabricated new fluorescence microscopy can take a one-time imaging for the 28×18mm dPCR gene chip with more than 20,000 multi micro-droplets within FAM, HEX, and ROX fluorescence channels. The optical system was designed with a numerical aperture (NA) of 0.106. Modulation transfer function (MTF) is higher than 0.675 at 70 lp/mm, and the function resolution capability is 10 µm with the whole magnification of -0.65times. The fly's eye lens-based illumination system was tested with a uniform output of over 90% in the whole ϕ34.7mm chip area. The design was tested, and the experimental results showed that this new system provides a fast, efficient, and professional optical imaging method for detection of the new emerged digital PCR gene chip, which has larger area and more channels.

Assessment of spawning site fidelity in interior Fraser River Coho salmon Oncorhynchus kisutch using otolith microchemistry, in British Columbia, Canada.

Coho Salmon Oncorhynchus kisutch show fidelity to natal spawning watersheds. Fine-scale homing, however, within rivers is not well understood. Interior Fraser Coho (IFC) salmon eggs were incubated at known spawning locations in the Coldwater River, two main stem sites and one-off channel pond site, providing otolith reference data for comparison to otolith signatures for returning adults using laser ablation inductively coupled plasma mass spectrometry. Elemental ratios for Ba:Ca and Sr:Ca in otoliths of juvenile O. kisutch differed significantly among the spawning locations examined. Juvenile otolith data were used to conduct a linear discriminant analysis to assess fine-scale homing in adults. Juvenile data were all assigned to the location where they had been incubated, producing a robust data set used to compare adult otoliths and define natal locations based on elemental signatures in otoliths of adult spawners. Homing and straying were apparent at the reach level; 57.1% of adults returned to their natal spawning locations, while 42.9% strayed to other spawning sites within the Coldwater River. Straying to novel incubation sites at the reach scale demonstrated plasticity in homing within a watershed.

Quantifying an overlooked aspect of partial migration using otolith microchemistry.

For the first time, an overlooked aspect of partial migration was quantified using otolith microchemistry and brown trout, Salmo trutta, as a model species. Relative contributions of freshwater resident and anadromous female brown trout to mixed-stock sea trout populations in the Baltic Sea were estimated. Out of 236 confirmed wild sea trout sampled around the coast of Estonia 88% were of anadromous maternal origin and 12% were of resident maternal origin. This novel finding underscores the importance of the resident contingent in maintaining the persistence and resilience of the migratory contingent.

Retracing migration pattern in reproductive and non-reproductive female kutum Rutilus frisii, in south Caspian Sea, using otolith microchemistry.

Because trace elements of otoliths are considered a natural marker capable of recognizing the chemical composition of ambient water and fish migration history, these elements could be potentially used to analyse the movement of reproductive (R) and non-reproductive (NR) mature-sized fish. Supposedly, it is not essential for NR individuals to migrate to rivers for spawning because they do not have developed gonads. To investigate the potential differences in migration history between female R and NR kutum, Rutilus frisii, in the southwest waters of the Caspian Sea, the ratios of Sr, Ba, Mg, Na, K and P to Ca in otoliths (from the core to the edge) were examined using laser ablation-inductively coupled plasma-mass spectrometry. In NR fish, a significant increase in Sr:Ca ratio in the otoliths' growth rings, likely due to greater seawater residency, and an increase in Ba:Ca ratio in the last two rings were observed. Increased Ba:Ca ratio could be due to the movement of NR mature-sized fish to the coastal zones for foraging. Seasonal physiological factors such as gonad maturation and spawning activity are more likely to be involved in differences in the other elemental ratios (Mg, Na, K and P). These results suggest that microchemical analyses of growth rings of otolith can be used as a valuable tool for better understanding the movement pattern of different types of adult fish, which could be completed with data from other methods like tagging.