Non-invasive iron deficiency diagnosis: a saliva-based approach using capillary flow microfluidics.

Iron deficiency anemia (IDA) is a condition characterized by lower-than-average iron (Fe) levels in the body, affecting a substantial number of young children and pregnant women globally. Existing diagnostic methods for IDA rely on invasive analysis of stored Fe in ferritin from blood samples, posing challenges, especially for toddlers and young children. To address this issue, saliva has been proposed as a non-invasive sample matrix for IDA diagnosis. However, conventional Fe analysis techniques often necessitate complex and costly instrumentation. This study presents the first non-invasive, saliva-based preliminary screening test for IDA using a nitrocellulose lateral flow system. In this study, we introduce a novel approach using the ferroin reaction with bathophenanthroline (Bphen) and ferrous (Fe2+) ions to quantify Fe levels in saliva. Our methodology involves a capillary flow-driven microfluidic device integrated into a lateral flow system utilizing nitrocellulose membranes. Here, we present the first instance of saliva on a nitrocellulose substrate to detect salivary Fe levels. The optimized system yielded a linear response over the 1-200 ppm range in buffer solution, with a limit of detection (LoD) of 5.6 ppm. Furthermore, the system demonstrated a linear response in pooled saliva samples across the 1-1000 ppm range, with a LoD of 55.1 ppm. These results underscore the potential of our capillary flow-driven microfluidic device as a viable non-invasive diagnostic tool for IDA, particularly in remote and resource-limited settings.

Speech Intelligibility in Patients with Facial Paralysis.

Background: Patients with facial paralysis often report frustration with communication; however, there are limited data regarding intelligibility deficiencies. Objective: To compare speech intelligibility in patients with severe and non-severe facial paralysis, and in patients with or without synkinesis. Methods: Video and audio data were reviewed retrospectively. Groups were stratified as follows: Group A - severe paralysis (Sunnybrook 0-20) without synkinesis; Group B - non-severe (Sunnybrook >20) paralysis without synkinesis; and Group C - non-severe paralysis with synkinesis. Intelligibility was assessed by lay-people and a speech and language pathologist (SLP) using the Frenchay Dysarthria Assessment Version 2 (FDA-2). A receiver operating characteristic (ROC) curve was used to determine a Sunnybrook cutoff for intelligibility. Results: Eighty cases were reviewed with mean age 55.6, 53.8% female. 25.0% were in Group A, 30.0% Group B, and 45.0% in Group C. Lay-people rated 15.0% and the SLP rated 28.7% as having intelligibility deficiency. An ROC curve demonstrated that patients with Sunnybrook ≤18.5 were more likely to have intelligibility abnormality. Conclusion: Patients with Sunnybrook ≤18.5 are more likely to demonstrate intelligibility deficiency. Clinicians with a more trained ear are more likely to identify intelligibility abnormality compared with lay-people. Those with synkinesis are more intelligible compared with those without it.

Pediatric scurvy case report: a novel presentation with deep vein thrombosis secondary to large bilateral spontaneous iliac subperiosteal hematomas.

BACKGROUND: Scurvy is an uncommon disease in developed countries caused by deficiency of vitamin C. We present a case of scurvy in a 14-year-old male with autism with both novel presentation and imaging findings. This case had the novel presentation of lower limb deep vein thrombosis (DVT) secondary to compression of the external iliac vein from large bilateral iliac wing subperiosteal hematomas. Subperiosteal hematoma is a well-recognised feature of scurvy but large and bilateral pelvic subperiosteal hematoma causing DVT has not previously been described. CASE PRESENTATION: A 14 year old Caucasian male with background of autism and severe dietary restriction presented with lower limb swelling and immobility. He was diagnosed with lower limb DVT. Further investigation revealed an iron deficiency anaemia, and he was found on MRI to have large bilateral subperiosteal iliac hematomata causing compression of the iliac vessels. He improved following treatment with vitamin C replacement and follow-up imaging demonstrated resolution of the DVT and hematoma. CONCLUSION: DVT is rare in children and when diagnosed should prompt investigation as to the underlying cause. This case demonstrates an unusual cause of DVT and as an unusual presentation of paediatric scurvy.

Improving design features and air bubble manipulation techniques for a single-step sandwich electrochemical ELISA incorporating commercial electrodes into capillary-flow driven immunoassay devices.

Integrating electrochemistry into capillary-flow driven immunoassay devices provides unique opportunities for quantitative point-of-care testing. Although custom electrodes can be inexpensive and are tunable, they require skilled fabrication. Here, we report the incorporation of a commercial electrode into a capillary-flow driven immunoassay (iceCaDI) device for a single end-user step sandwich electrochemical enzyme-linked immunosorbent assay (ELISA). The iceCaDI device is a pump-free portable microfluidic device with an integrated commercial screen-printed electrode and flow driven by capillary action. The iceCaDI device is composed of alternating polyester transparency film and double-sided adhesive film layers that are patterned with a laser cutter. This platform was designed to address known limitations of laminated device fabrication methods and operation. First, we developed a foldable laminated device fabrication using hinges for easy assembly and precise alignment. Second, reagent dispersing was achieved by incorporating a 1 mm wide arrow-shaped notch in the middle of the channel that trapped an air bubble and formed a baffle that facilitated reagent spreading to cover the detection area. Third, small vent holes were added to the top layer of the channels to prevent air bubbles from blocking flow. Finally, we fabricated a CRP immunosensor with a detection range of 0.625 to 10.0 µg mL-1 as a proof-of-concept to demonstrate an automatically driven sandwich electrochemical ELISA using the iceCaDI device. Three concentrations of CRP were successfully measured under flow conditions within 8 min. Our proposed device is a promising approach and a step forward in the development of point-of-care (POC) devices for techniques that traditionally require multiple user steps.

Recent Trends in Nanomaterial Based Electrochemical Sensors for Drug Detection: Considering Green Assessment.

An individual's therapeutic drug exposure level is directly linked to corresponding clinical effects. Rapid, sensitive, inexpensive, portable and reliable devices are needed for diagnosis related to drug exposure, treatment, and prognosis of diseases. Electrochemical sensors are useful for drug monitoring due to their high sensitivity and fast response time. Also, they can be combined with portable signal read-out devices for point-of-care applications. In recent years, nanomaterials such as carbon-based, carbon-metal nanocomposites, noble nanomaterials have been widely used to modify electrode surfaces due to their outstanding features including catalytic abilities, conductivity, chemical stability, biocompatibility for development of electrochemical sensors. This review paper presents the most recent advances about nanomaterials-based electrochemical sensors including the use of green assessment approach for detection of drugs including anticancer, antiviral, anti-inflammatory, and antibiotics covering the period from 2019 to 2023. The sensor characteristics such as analyte interactions, fabrication, sensitivity, and selectivity are also discussed. In addition, the current challenges and potential future directions of the field are highlighted.

Multiplexed Capillary-Flow Driven Immunoassay for Respiratory Illnesses.

Multiplexed analysis in medical diagnostics is widely accepted as a more thorough and complete method compared to single-analyte detection. While analytical methods like polymerase chain reaction and enzyme-linked immunosorbent assay (ELISA) exist for multiplexed detection of biomarkers, they remain time-consuming and expensive. Lateral flow assays (LFAs) are an attractive option for point-of-care testing, and examples of multiplexed LFAs exist. However, these devices are limited by spatial resolution of test lines, large sample volume requirements, cross-reactivity, and poor sensitivity. Recent work has developed capillary-flow microfluidic ELISA platforms as a more sensitive alternative to LFAs; however, multiplexed detection on these types of devices has yet to be demonstrated. In the aftermath of the initial SARS-CoV-2 pandemic, the need for rapid, sensitive point-of-care devices has become ever clearer. Moving forward, devices that can distinguish between diseases with similar presenting symptoms would be the ideal home diagnostic. Here, the first example of a multiplexed capillary-flow immunoassay device for the simultaneous detection of multiple biomarkers is reported. From a single sample addition step, the reagents and washing steps required for two simultaneous ELISAs are delivered to spatially separated test strips. Visual results can be obtained in <15 min, and images captured with a smartphone can be analyzed for quantitative data. This device was used to distinguish between and quantify H1N1 hemagglutinin (HA) and SARS-CoV-2 nucleocapsid protein (N-protein). Using this device, analytical detection limits of 840 and 133 pg/mL were obtained for hemagglutinin and nucleocapsid protein, respectively. The presence of one target in the device did not increase the signal on the other test line, indicating no cross-reactivity between the assays. Additionally, simultaneous detection of both N-protein and HA was performed as well as simultaneous detection of N-protein and human C-reactive protein (CRP). Elevated levels of CRP in a patient infected with SARS-CoV-2 have been shown to correlate with more severe outcomes and a greater risk of death as well. To further expand on the simultaneous detection of two biomarkers, CRP and N-protein were detected simultaneously, and the presence of SARS-CoV-2 N-protein did not interfere with the detection of CRP when both targets were present in the sample.

Microfluidics in environmental analysis: advancements, challenges, and future prospects for rapid and efficient monitoring.

Microfluidic devices have emerged as advantageous tools for detecting environmental contaminants due to their portability, ease of use, cost-effectiveness, and rapid response capabilities. These devices have wide-ranging applications in environmental monitoring of air, water, and soil matrices, and have also been applied to agricultural monitoring. Although several previous reviews have explored microfluidic devices' utility, this paper presents an up-to-date account of the latest advancements in this field for environmental monitoring, looking back at the past five years. In this review, we discuss devices for prominent contaminants such as heavy metals, pesticides, nutrients, microorganisms, per- and polyfluoroalkyl substances (PFAS), etc. We cover numerous detection methods (electrochemical, colorimetric, fluorescent, etc.) and critically assess the current state of microfluidic devices for environmental monitoring, highlighting both their successes and limitations. Moreover, we propose potential strategies to mitigate these limitations and offer valuable insights into future research and development directions.

Gene pyramiding for boosted plant growth and broad abiotic stress tolerance.

Abiotic stresses such as salinity, heat and drought seriously impair plant growth and development, causing a significant loss in crop yield and ornamental value. Biotechnology approaches manipulating specific genes prove to be effective strategies in crop trait modification. The Arabidopsis vacuolar pyrophosphatase gene AVP1, the rice SUMO E3 ligase gene OsSIZ1 and the cyanobacterium flavodoxin gene Fld have previously been implicated in regulating plant stress responses and conferring enhanced tolerance to different abiotic stresses when individually overexpressed in various plant species. We have explored the feasibility of combining multiple favourable traits brought by individual genes to acquire superior plant performance. To this end, we have simultaneously introduced AVP1, OsSIZ1 and Fld in creeping bentgrass. Transgenic (TG) plants overexpressing these three genes performed significantly better than wild type controls and the TGs expressing individual genes under both normal and various abiotic stress conditions, exhibited significantly enhanced plant growth and tolerance to drought, salinity and heat stresses as well as nitrogen and phosphate starvation, which were associated with altered physiological and biochemical characteristics and delicately fine-tuned expression of genes involved in plant stress responses. Our results suggest that AVP1, OsSIZ1 and Fld function synergistically to regulate plant development and plant stress response, leading to superior overall performance under both normal and adverse environments. The information obtained provides new insights into gene stacking as an effective approach for plant genetic engineering. A similar strategy can be extended for the use of other beneficial genes in various crop species for trait modifications, enhancing agricultural production.

Characterization of Factors Affecting Stripping Voltammetry on Thermoplastic Electrodes.

Thermoplastic carbon electrodes (TPEs) are an alternative form of carbon composite electrodes that have shown excellent electrochemical performance with applications in biological sensing. However, little has been done to apply TPEs to environmental sensing, specifically heavy metal analysis. The work here focuses on lead analysis and based on their electrochemical properties, TPEs are expected to outperform other carbon composite materials; however, despite testing multiple formulations, TPEs showed inferior performance. Detailed electrode characterization was conducted to examine the cause for poor lead sensing behavior. X-Ray photoelectron spectroscopy (XPS) was used to analyze the surface functional groups, indicating that acidic and alkaline functional groups impact lead electrodeposition. Further, scanning electron microscopy (SEM) and electrochemical characterization demonstrated that both the binder and graphite can influence the surface morphology, electroactive area, and electron kinetics.

Surface Modification of Thermoplastic Electrodes for Biosensing Applications via Copper-Catalyzed Click Chemistry.

Cu(I)-catalyzed 1,3-dipolar cycloaddition (CuAAC), also known as click chemistry, has been demonstrated to be highly robust while providing versatile surface chemistry. One specific application is biosensor fabrication. Recently, we developed thermoplastic electrodes (TPEs) as an alternative to traditional carbon composite electrodes in terms of cost, performance, and robustness. However, their applications in biosensing are currently limited due to a lack of facile methods for electrode modification. Here, we demonstrate the feasibility of using CuAAC following the diazonium grafting of TPEs to take advantage of two powerful technologies for developing a customizable and versatile biosensing platform. After a stepwise characterization of the electrode modification procedures was performed, electrodes were modified with model affinity reagents. Streptavidin and streptavidin-conjugated IgG antibodies were successfully immobilized on the TPE surface, as confirmed by electrochemical impedance spectroscopy and X-ray photoelectron spectroscopy.

Maximizing flow rate in single paper layer, rapid flow microfluidic paper-based analytical devices.

Small, single-layer microfluidic paper-based analytical devices (µPADs) offer potential for a range of point-of-care applications; however, they have been limited to low flow rates. Here, we investigate the role of laser cutting paper channels in maximizing flow rate in small profile devices with limited fluid volumes. We demonstrate that branching, laser-cut grooves can provide a 59.23-73.98% improvement in flow rate over a single cut, and a 435% increase over paper alone. These design considerations can be applied to more complex microfluidic devices with the aim of increasing the flow rate, and could be used in stand-alone channels for self-pumping. Supplementary Information: The online version contains supplementary material available at 10.1007/s10404-023-02679-8.

Microfluidic organotypic device to test intestinal mucosal barrier permeability ex vivo.

To protect the body from external pathogens, the intestines have sophisticated epithelial and mucosal barriers. Disruptions to barrier integrity are associated with a variety of disorders such as irritable bowel disease, Crohn's disease, and celiac disease. One critical component of all barriers are collagens in the extracellular matrix. While the importance of the intestinal barrier is established, current models lack the ability to represent the complex biology that occurs at these barriers. For the current study a microfluidic device model was modified to determine the effectiveness of collagen breakdown to cause barrier disruption. Bacterial collagenase was added for 48 h to the luminal channel of a dual flow microfluidic device to examine changes in intestinal barrier integrity. Tissues exhibited dose-dependent alterations in immunoreactive collagen-1 and claudin-1, and coincident disruption of the epithelial monolayer barrier as indicated by goblet cell morphologies. This ex vivo model system offers promise for further studies exploring factors that affect gut barrier integrity and potential downstream consequences that cannot be studied in current models.

Capillary flow-driven immunoassay platform for COVID-19 antigen diagnostics.

Over the last few years, the SARS-CoV-2 pandemic has made the need for rapid, affordable diagnostics more compelling than ever. While traditional laboratory diagnostics like PCR and well-plate ELISA are sensitive and specific, they can be costly and take hours to complete. Diagnostic tests that can be used at the point-of-care or at home, like lateral flow assays (LFAs) are a simple, rapid alternative, but many commercially available LFAs have been criticized for their lack of sensitivity compared to laboratory methods like well-plate ELISAs. The Capillary-Driven Immunoassay (CaDI) device described in this work uses microfluidic channels and capillary action to passively automate the steps of a traditional well-plate ELISA for visual read out. This work builds on prior capillary-flow devices by further simplifying operation and use of colorimetric detection. Upon adding sample, an enzyme-conjugated secondary antibody, wash steps, and substrate are sequentially delivered to test and control lines on a nitrocellulose strip generating a colorimetric response. The end user can visually detect SARS-CoV-2 antigen in 15-20 min by naked eye, or results can be quantified using a smartphone and software such as ImageJ. An analytical detection limit of 83 PFU/mL for SARS-CoV-2 was determined for virus in buffer, and 222 PFU/mL for virus spiked into nasal swabs using image analysis, similar to the LODs determined by traditional well-plate ELISA. Additionally, a visual detection limit of 100 PFU/mL was determined in contrived nasal swab samples by polling 20 untrained end-users. While the CaDI device was used for detecting clinically relevant levels of SARS-CoV-2 in this study, the CaDI device can be easily adapted to other immunoassay applications by changing the reagents and antibodies.

Why fake death? Environmental and genetic control of tonic immobility in larval lacewings (Neuroptera: Chrysopidae).

Tonic immobility is a passive antipredator strategy employed late in the predation sequence that may decrease individual mortality in prey animals. Here, we investigate how energetic state and genetic predisposition influence antipredator decision-making in green lacewing larvae, Chrysoperla plorabunda (Fitch), using simulated predatory encounters. We demonstrate that tonic immobility is a plastic response influenced by energetic resource limitation. Larvae exposed to 1 or 2 days of food deprivation initiate tonic immobility more often and with less physical provocation than individuals fed ad libitum. Recently molted individuals exposed to food deprivation, the individuals most energetically challenged, engage in tonic immobility at a higher rate than any other group. We also find that variation in antipredator strategy between individuals is partly the result of within-population genetic variation. We estimate the propensity to enter tonic immobility to have a broad-sense heritability of 0.502. Taken together our results suggest that larval lacewings under energetic stress are more likely to engage in tonic immobility. Yet, energetic state does not explain all within-population variation, as individuals can have a genetic predisposition for tonic immobility.

Fast and easy synthesis of silver, copper, and bimetallic nanoparticles on cellulose paper assisted by ultrasound.

This work focuses on a systematic method to produce Ag, Cu, and Ag/Cu metallic nanoparticles (MNPs) in situ assisted with ultrasound on cellulose paper. By tuning the concentration of AgNO3 and CuSO4 salt precursors and ultrasound time, combined with a fixed concentration of ascorbic acid (AA) as a reducing agent, it was possible to control the size, morphology, and polydispersity of the resulting MNPs on cellulose papers. Notably, high yield and low polydispersity of MNPs and bimetallic nanoparticles are achieved by increasing the sonication time on paper samples pre-treated with salt precursors before reduction with AA. Moreover, mechanical analysis on paper samples presenting well-dispersed and distributed MNPs showed slightly decreasing values of Young's modulus compared to neat papers. The strain at break is substantially improved in papers containing solely Ag or Cu MNPs. The latter suggests that the elastic/plastic transition and deformation of papers are tuned by cellulose and MNPs interfacial interaction, as indicated by mechanical analysis. The proposed method provides insights into each factor affecting the sonochemistry in situ synthesis of MNPs on cellulose papers. In addition, it offers a straightforward alternative to scale up the production of MNPs on paper, ensuring an eco-friendly method.

Application of Formulation Principles to Stability Issues Encountered During Processing, Manufacturing, and Storage of Drug Substance and Drug Product Protein Therapeutics.

The field of formulation and stabilization of protein therapeutics has become rather extensive. However, most of the focus has been on stabilization of the final drug product. Yet, proteins experience stress and degradation through the manufacturing process, starting with fermentaition. This review describes how formulation principles can be applied to stabilize biopharmaceutical proteins during bioprocessing and manufacturing, considering each unit operation involved in prepration of the drug substance. In addition, the impact of the container on stabilty is discussed as well.

Recent Advances of Optical Biosensors in Veterinary Medicine: Moving Towards the Point of Care Applications.

While food safety issues are attracting public concern due to their detrimental effects on human health, monitoring livestock health is urgently needed to diagnose animal diseases at an early stage by applying proper treatments, controlling, and preventing outbreaks, particularly in resource- limited countries. In addition, unhealthy farms are not only a threat to livestock but also to human lives. The available diagnostic techniques for the detection of key health threats within both the food and livestock sectors require labor-intensive and time-consuming experimental procedures and sophisticated and expensive instruments. To tackle this issue, optical biosensing strategies have been incorporated into point-of-care (POC) systems, offering real-time monitoring, field-deployable, and low-cost devices, which help make on-the-spot decisions. This review aims to discuss the recent cutting-edge research on POC optical biosensing platforms for on-farm diagnosis of animal diseases and on-site detection of animal-derived food-borne contaminants, including pathogens, antibiotics, and mycotoxins. Moreover, this review briefly presents the basic knowledge of various types of optical biosensors and their development using various recent strategies, including nanomaterial combinations, to enhance their performance in POC tests. This review is expected to help scientists to understand the evolution and challenges in the development of point-of-care biosensors for the food and livestock industry, benefiting global healthcare.

Microfluidic paper-based analytical devices for simultaneous detection of oxidative potential and copper in aerosol samples.

The potential reach of point-of-care (POC) diagnostics into daily routines for exposure to reactive oxygen species (ROS) and Cu in aerosolized particulate matter (PM) demands that microfluidic paper-based analytical devices (µPADs) take into consideration the simple detection of these toxic PM components. Here, we propose µPADs with a dual-detection system for simultaneous ROS and Cu(II) detection. For colorimetric ROS detection, the glutathione (GSH) assay with a folding design to delay the reaction yielded complete ROS and GSH oxidation, and improved homogeneity of color development relative to using the lateral flow pattern. For electrochemical Cu(II) determination, 1,10-phenanthroline/Nafion modified graphene screen-printed electrodes showed ability to detect Cu(II) down to pg level being low enough to be applied to PM analysis. No intra- and inter-interference affecting both systems were found. The proposed µPADs obtained LODs for 1,4-naphthoquinone (1,4-NQ), used as the ROS representative, and Cu(II) of 8.3 ng and 3.6 pg, respectively and linear working ranges of 20 to 500 ng for ROS and 1 × 10-2 to 2 × 102 ng for Cu(II). Recovery of the method was between 81.4 and 108.3% for ROS and 80.5-105.3% for Cu(II). Finally, the sensors were utilized for simultaneous ROS and Cu(II) determination in PM samples and the results statistically agreed with those using the conventional methods at 95% confidence.

Large-scale fabrication of ion-selective electrodes for simultaneous detection of Na+, K+, and Ca2+ in biofluids using a smartphone-based potentiometric sensing platform.

A significant bottleneck exists for mass-production of ion-selective electrodes despite recent developments in manufacturing technologies. Here, we present a fully-automated system for large-scale production of ISEs. Three materials, including polyvinyl chloride, polyethylene terephthalate and polyimide, were used as substrates for fabricating ion-selective electrodes (ISEs) using stencil printing, screen-printing and laser engraving, respectively. We compared sensitivities of the ISEs to determine the best material for the fabrication process of the ISEs. The electrode surfaces were modified with various carbon nanomaterials including multi-walled carbon nanotubes, graphene, carbon black, and their mixed suspensions as the intermediate layer to enhance sensitivities of the electrodes. An automated 3D-printed robot was used for the drop-cast procedure during ISE fabrication to eliminate manual steps. The sensor array was optimized, and the detection limits were 10-5 M, 10-5 M and 10-4 M for detection of K+, Na+ and Ca2+ ions, respectively. The sensor array integrated with a portable wireless potentiometer was used to detect K+, Na+ and Ca2+ in real urine and simulated sweat samples and results obtained were in agreement with ICP-OES with good recoveries. The developed sensing platform offers low-cost detection of electrolytes for point-of-care applications.

Co-Clinical Imaging Metadata Information (CIMI) for Cancer Research to Promote Open Science, Standardization, and Reproducibility in Preclinical Imaging.

Preclinical imaging is a critical component in translational research with significant complexities in workflow and site differences in deployment. Importantly, the National Cancer Institute's (NCI) precision medicine initiative emphasizes the use of translational co-clinical oncology models to address the biological and molecular bases of cancer prevention and treatment. The use of oncology models, such as patient-derived tumor xenografts (PDX) and genetically engineered mouse models (GEMMs), has ushered in an era of co-clinical trials by which preclinical studies can inform clinical trials and protocols, thus bridging the translational divide in cancer research. Similarly, preclinical imaging fills a translational gap as an enabling technology for translational imaging research. Unlike clinical imaging, where equipment manufacturers strive to meet standards in practice at clinical sites, standards are neither fully developed nor implemented in preclinical imaging. This fundamentally limits the collection and reporting of metadata to qualify preclinical imaging studies, thereby hindering open science and impacting the reproducibility of co-clinical imaging research. To begin to address these issues, the NCI co-clinical imaging research program (CIRP) conducted a survey to identify metadata requirements for reproducible quantitative co-clinical imaging. The enclosed consensus-based report summarizes co-clinical imaging metadata information (CIMI) to support quantitative co-clinical imaging research with broad implications for capturing co-clinical data, enabling interoperability and data sharing, as well as potentially leading to updates to the preclinical Digital Imaging and Communications in Medicine (DICOM) standard.