Filtered Saliva for Rapid and Accurate Analyte Detection for POC Diagnostics.

Saliva has shown considerable promise as a diagnostic medium for point-of-care (POC) and over-the-counter (OTC) diagnostic devices due to the non-invasive nature of its collection. However, a significant limitation of saliva-based detection is undesirable interference in a sensor's readout caused by interfering components in saliva. In this study, we develop standardized sample treatment procedures to eliminate bubbles and interfering molecules while preserving the sample's target molecules such as spike (S) protein and glucose. We then test the compatibility of the pretreatment system with our previously designed SARS-CoV-2 and glucose diagnostic biosensing systems for detecting S protein and glucose in subject saliva. Ultimately, the effectiveness of each filter in enhancing biomarker sensitivity is assessed. The results show that a 20 mg nylon wool (NW) filter shows an 80% change in viscosity reduction with only a 6% reduction in protein content, making it an appropriate filter for the salivary S protein diagnostic system. Meanwhile, a 30 mg cotton wool (CW) filter is identified as the optimal choice for salivary glucose detection, achieving a 90% change in viscosity reduction and a 60.7% reduction in protein content with a minimal 4.3% reduction in glucose content. The NW pretreatment filtration significantly improves the limit of detection (LOD) for salivary S protein detection by five times (from 0.5 nM to 0.1 nM) and it reduces the relative standard deviation (RSD) two times compared to unfiltered saliva. Conversely, the CW filter used for salivary glucose detection demonstrated improved linearity with an R2 of 0.99 and a sensitivity of 36.6 µA/mM·cm2, over twice as high as unfiltered saliva. This unique filtration process can be extended to any POC diagnostic system and optimized for any biomarker detection, making electrochemical POC diagnostics more viable in the current market.

Crystallographic control of the fabrication of an extremely sophisticated shell surface microornament in the glass scallop Catillopecten.

The external surface microornament of the glass scallops Catillopecten natalyae and malyutinae is made by calcitic spiny projections consisting of a stem that later divides into three equally spaced and inclined branches (here called aerials). C. natalyae contains larger and smaller aerials, whereas C. malyutinae only secreted aerials of the second type. A remarkable feature is that aerials within each type are fairly similar in size and shape and highly co-oriented, thus constituting a most sophisticated microornament. We demonstrate that aerials are single crystals whose morphology is strongly controlled by the crystallography, with the stem being parallel to the c-axis of calcite, and the branches extending along the edges of the {104} calcite rhombohedron. They grow epitaxially onto the foliated prisms of the outer shell layer. The co-orientation of the prisms explains that of the aerials. We have developed a model in which every aerial grows within a periostracal pouch. When this pouch reaches the growth margin, the mantle initiates the production of the aerial. Nevertheless, later growth of the aerial is remote, i.e. far from the contact with the mantle. We show how such an extremely sophisticated microornament has a morphology and co-orientation which are determined by crystal growth.

A Rapid Label-Free Disposable Electrochemical Salivary Point-of-Care Sensor for SARS-CoV-2 Detection and Quantification.

The coronavirus disease 2019 (COVID-19) pandemic has created an urgent need for accurate early diagnosis and monitoring. A label-free rapid electrochemical point-of-care (POC) biosensor for SARS-CoV-2 detection in human saliva is reported here to help address the shortcomings of traditional nucleic acid amplification methods and give a quantitative assessment of the viral load to track infection status anywhere, using disposable electrochemical sensor chips. A new chemical construct of gold nanoparticles (GNp) and thionine (Th) are immobilized on carboxylic acid functionalized carbon nanotubes (SWCNT-COOH) for high-performance biosensing. The sensor uses saliva with a one-step pretreatment and simple testing procedure as an analytical medium due to the user-friendly and non-invasive nature of its procurement from patients. The sensor has a response time of 5 min with a limit of detection (LOD) reaching 200 and 500 pM for the freely suspended spike (S) protein in phosphate buffer saline (PBS) and human saliva, respectively. The sensor's performance was also proven for detecting a COVID-19 pseudovirus in an electrolyte solution with a LOD of 106 copies/mL. The results demonstrate that the optimized POC sensor developed in this work is a promising device for the label-free electrochemical biosensing detection of SARS-CoV-2 and different species of viruses.

The argonaut constructs its shell via physical self-organization and coordinated cell sensorial activity.

The shell of the cephalopod Argonauta consists of two layers of fibers that elongate perpendicular to the shell surfaces. Fibers have a high-Mg calcitic core sheathed by thin organic membranes (>100 nm) and configurate a polygonal network in cross section. Their evolution has been studied by serial sectioning with electron microscopy-associated techniques. During growth, fibers with small cross-sectional areas shrink, whereas those with large sections widen. It is proposed that fibers evolve as an emulsion between the fluid precursors of both the mineral and organic phases. When polygons reach big cross-sectional areas, they become subdivided by new membranes. To explain both the continuation of the pattern and the subdivision process, the living cells from the mineralizing tissue must perform contact recognition of the previously formed pattern and subsequent secretion at sub-micron scale. Accordingly, the fabrication of the argonaut shell proceeds by physical self-organization together with direct cellular activity.

Geographical variability and parasitism on body size, reproduction and shell characteristics of the keyhole limpet Fissurella crassa (Mollusca: Vetigastropoda).

Environmental variation may alter biological interactions and their ecological consequences. For instance, in marine ecosystems hosts and parasites are subject to environmental variability across latitudinal gradients, and their co-evolutionary dynamics may be the result of the interplay with local physical-chemical variables in seawater. Thus, assessing the environmental conditions required for a host in order to improve their survival is essential to understand the host-parasite interaction and dynamics. In this study, we evaluated the impact of parasitism by Proctoeces humboldti on the body size and reproduction of the intertidal keyhole limpet Fissurella crassa collected from three populations spanning ca. 1500 km along the latitudinal gradient of the Chilean coast. In addition, for the first time, we explore whether the effect of parasitism can be extended to changes in the organic composition and mechanical properties of the host shell. Our results show that parasitism prevalence and intensity, and body size of F. crassa increased in central Chile (ca. 33°S). Unlike body size, which was greater in parasitized limpets than in non-parasitized limpets at the three study sites, reproductive performance followed this trend only in central Chile populations, with no differences between parasitized and non-parasitized limpets collected in the northern Chilean (ca. 23°S), and lower in parasitized than non-parasitized individuals from the south-central Chile (ca. 37°S). The organic composition of F. crassa shells showed significant differences between parasite conditions (e.g. polysaccharides and water decreased in parasitized limpets) and across sites (e.g. proteins levels increase in shell of parasitized limpets from central Chile, but decreased at south-central Chile). However, variability in shell mechanical properties (e.g. toughness and elastic module) do not showed significant differences across sites and parasitism condition. These results suggest the interplay of both parasitism and environmental fluctuations upon the reproductive performance and morphology of the host. In addition, our result highlight that the host may also trade-offs reproduction, growth and shell organic composition to maintain the shell functionality (e.g. protection for mechanical forces and durophagous predators).

Upwelling intensity modulates the fitness and physiological performance of coastal species: Implications for the aquaculture of the scallop Argopecten purpuratus in the Humboldt Current System.

Understanding how marine species cope with the natural environmental variability of their native habitats will provide significant information about their sensitivity to the potential environmental changes driven by climate change. In particular, marine species inhabiting upwelling ecosystems are experiencing low seawater temperatures, as well as, acidic and low oxygen conditions as a consequence of the nature of the deep upwelled waters. Our study is focused on one of the most important socio-economical resources of the Humboldt Current System (HCS): the scallop Argopecten purpuratus which has been historically subjected to intensive aquaculture in areas influenced by upwelling processes. Here, a long-term field experiment was performed to understand how tolerant and well-locally-adapted is A. purpuratus to upwelling conditions by studying a set of fitness, physiological, and biomineralogical traits. Stronger upwelling generated a minor water column stratification, with lower temperatures, pH, and oxygen conditions. On the contrary, as upwelling weakened, temperature, pH, and oxygen availability increased. Finally, upwelling intensity also determined the number, duration, and intensity of the cooling and de-oxygenation events occurring in A. purpuratus habitat, as well as, the food availability (chlorophyll-a concentration, Chl-a). Physiologically, A. purpuratus was able to cope with stressful environmental conditions imposed by higher upwelling intensities by enhancing its metabolic and calcification rates, as well, producing higher concentrations of the shell organic matter. These physiological changes impacted the total energy budget, which was highly dependent on Chl-a concentration, and revealed important traits trade-offs with significant fitness costs (higher mortalities emerged when longer and more intense upwelling events succeed). Our study increases the knowledge about the physiological performance and tolerance of this important resource to the ocean acidification and ocean-deoxygenation imposed by variable upwelling intensities, as well as, its potential vulnerability under future changing conditions driven by a potential upwelling intensification.

The combined effects of salinity and pH on shell biomineralization of the edible mussel Mytilus chilensis.

Coastal ecosystems influenced by river discharges are subjected to important environmental changes. Understanding how marine biota cope with its environment is relevant in predicting the responses to future conditions imposed by climate change. To date, a large number of studies have addressed the role of pH on shell and biomineralization properties on multiple calcifying species; however the role of salinity in combination with other stressors has been poorly studied. In particular, the edible mussel Mytilus chilensis, an important marine resource of the Chilean coasts, inhabits estuarine areas which show high natural variability in terms of pH and salinity. Here, we studied how M. chilensis shell periostracum, shell organic matrix and crystal orientation are affected by different pH (8.1 and 7.7) and salinity conditions (30, 25 and 20 psu), isolated and in combination, at different time intervals. Our results show differences in the plasticity of the different biomineralogical properties studied during the experiment under the different pH and salinity treatments. While the periostracum thickness and the total shell organic matter were not affected by pH and salinity, the periostracum organic composition did. Higher amounts of polysaccharides were observed under low pH conditions after 20 days of experiment, while after 60 days, low salinity was responsible for the decrease of the polysaccharides and proteins in the periostracum. Low salinity also produced a major disorder in crystal organization at the outer shell surface. Finally, total shell weight was only affected by low pH conditions under lower salinity conditions (20 psu). From the results, in the majority of the shell properties observed we did not observe any combined effect of pH and salinity. Also, we detected that the magnitude of the impacts of salinity and pH are variable and time-dependent. This would be suggesting some level of acclimatization of M. chilensis to lower pH and salinity conditions.

Articulation and growth of skeletal elements in balanid barnacles (Balanidae, Balanomorpha, Cirripedia).

The morphology and ultrastructure of the shells of two balanid species have been examined, paying special attention to the three types of boundaries between plates: (i) radii-parietes, (ii) alae-sheaths, and (iii) parietes-basal plate. At the carinal surfaces of the radii and at the rostral surfaces of the alae, there are series of crenulations with dendritic edges. The crenulations of the radius margins interlock with less prominent features of the opposing paries margins, whereas the surfaces of the longitudinal abutments opposing the ala margins are particularly smooth. The primary septa of the parietes also develop dendritic edges, which abut the internal surfaces of the primary tubes of the base plates. In all cases, there are chitino-proteinaceous organic membranes between the abutting structures. Our observations indicate that the very edges of the crenulations and the primary septa are permanently in contact with the organic membranes. We conclude that, when a new growth increment is going to be produced, the edges of both the crenulations and the primary septa pull the viscoelastic organic membranes locally, with the consequent formation of viscous fingers. For the abutting edges to grow, calcium carbonate must diffuse across the organic membranes, but it is not clear how growth of the organic membranes themselves is accomplished, in the absence of any cellular tissue.