Advancements in Cortisol Detection: From Conventional Methods to Next-Generation Technologies for Enhanced Hormone Monitoring.

The hormone cortisol, released as the end-product of the hypothalamic-pituitary-adrenal (HPA) axis, has a well-characterized circadian rhythm that enables an allostatic response to external stressors. When the pattern of secretion is disrupted, cortisol levels are chronically elevated, contributing to diseases such as heart attacks, strokes, mental health disorders, and diabetes. The diagnosis of chronic stress and stress related disorders depends upon accurate measurement of cortisol levels; currently, it is quantified using mass spectroscopy or immunoassay, in specialized laboratories with trained personnel. However, these methods are time-consuming, expensive and are unable to capture the dynamic biorhythm of the hormone. This critical review traces the path of cortisol detection from traditional laboratory-based methods to decentralised cortisol monitoring biosensors. A complete picture of cortisol biology and pathophysiology is provided, and the importance of precision medicine style monitoring of cortisol is highlighted. Antibody-based immunoassays still dominate the pipeline of development of point-of-care biosensors; new capture molecules such as aptamers and molecularly imprinted polymers (MIPs) combined with technologies such as microfluidics, wearable electronics, and quantum dots offer improvements to limit of detection (LoD), specificity, and a shift toward rapid or continuous measurements. While a variety of different sensors and devices have been proposed, there still exists a need to produce quantitative tests for cortisol ─ using either rapid or continuous monitoring devices that can enable a personalized medicine approach to stress management. This can be addressed by synergistic combinations of technologies that can leverage low sample volumes, relevant limit of detection and rapid testing time, to better account for cortisol's shifting biorhythm. Trends in cortisol diagnostics toward rapid and continuous monitoring of hormones are highlighted, along with insights into choice of sample matrix.

Time- and distance-resolved robotic imaging of fluid flow in vertical microfluidic strips: a new technique for quantitative, multiparameter measurement of global haemostasis.

Measuring the complex processes of blood coagulation, haemostasis and thrombosis that are central to cardiovascular health and disease typically requires a choice between high-resolution low-throughput laboratory assays, or simpler less quantitative tests. We propose combining mass-produced microfluidic devices with open-source robotic instrumentation to enable rapid development of affordable and portable, yet high-throughput and performance haematological testing. A time- and distance-resolved fluid flow analysis by Raspberry Pi imaging integrated with controlled sample addition and illumination, enabled simultaneous tracking of capillary rise in 120 individual capillaries (∼160, 200 or 270 µm internal diameter), in 12 parallel disposable devices. We found time-resolved tracking of capillary rise in each individual microcapillary provides quantitative information about fluid properties and most importantly enables quantitation of dynamic changes in these properties following stimulation. Fluid properties were derived from flow kinetics using a pressure balance model validated with glycerol-water mixtures and blood components. Time-resolved imaging revealed fluid properties that were harder to determine from a single endpoint image or equilibrium analysis alone. Surprisingly, instantaneous superficial fluid velocity during capillary rise was found to be largely independent of capillary diameter at initial time points. We tested if blood function could be measured dynamically by stimulating blood with thrombin to trigger activation of global haemostasis. Thrombin stimulation slowed vertical fluid velocity consistent with a dynamic increase in viscosity. The dynamics were concentration-dependent, with highest doses reducing flow velocity faster (within 10 s) than lower doses (10-30 s). This open-source imaging instrumentation expands the capability of affordable microfluidic devices for haematological testing, towards high-throughput multi-parameter blood analysis needed to understand and improve cardiovascular health.

Moving microcapillary antibiotic susceptibility testing (mcAST) towards the clinic: unravelling kinetics of detection of uropathogenic E. coli, mass-manufacturing and usability for detection of urinary tract infections in human urine.

Innovation in infection based point-of-care (PoC) diagnostics is vital to avoid unnecessary use of antibiotics and the development of antimicrobial resistance. Several groups including our research team have in recent years successfully miniaturised phenotypic antibiotic susceptibility tests (AST) of isolated bacterial strains, providing validation that miniaturised AST can match conventional microbiological methods. Some studies have also shown the feasibility of direct testing (without isolation or purification), specifically for urinary tract infections, paving the way for direct microfluidic AST systems at PoC. As rate of bacteria growth is intrinsically linked to the temperature of incubation, transferring miniaturised AST nearer the patient requires building new capabilities in terms of temperature control at PoC, furthermore widespread clinical use will require mass-manufacturing of microfluidic test strips and direct testing of urine samples. This study shows for the first-time application of microcapillary antibiotic susceptibility testing (mcAST) directly from clinical samples, using minimal equipment and simple liquid handling, and with kinetics of growth recorded using a smartphone camera. A complete PoC-mcAST system was presented and tested using 12 clinical samples sent to a clinical laboratory for microbiological analysis. The test showed 100% accuracy for determining bacteria in urine above the clinical threshold (5 out of 12 positive) and achieved 95% categorical agreement for 5 positive urines tested with 4 antibiotics (nitrofurantoin, ciprofloxacin, trimethoprim and cephalexin) within 6 h compared to the reference standard overnight AST method. A kinetic model is presented for metabolization of resazurin, demonstrating kinetics of degradation of resazurin in microcapillaries follow those observed for a microtiter plate, with time for AST dependent on the initial CFU ml-1 of uropathogenic bacteria in the urine sample. In addition, we show for the first time that use of air-drying for mass-manufacturing and deposition of AST reagents within the inner surface of mcAST strips matches results obtained with standard AST methods. These results take mcAST a step closer to clinical application, for example as PoC support for antibiotic prescription decisions within a day.

Label-free 1D microfluidic dipstick counting of microbial colonies and bacteriophage plaques.

Counting viable bacterial cells and functional bacteriophage is fundamental to microbiology underpinning research, surveillance, biopharmaceuticals and diagnostics. Colony forming unit (CFU) and plaque forming unit (PFU) counting still requires slow and laborious solid culture on agar in Petri dishes or plates. Here, we show that dip-stick microfluidic strips can be used without growth indicator dye for rapid and simple CFU ml-1 and PFU ml-1 measurement. We demonstrate for the first time that fluoropolymer microcapillaries combined with digital imaging allow bacteriophage plaques to be counted rapidly in a dip-and-test format. The microfluidic length scales offer a linear 1-dimensional alternative to a 2D solid agar medium surface, with colonies or plaques clearly visible as "dashes" or "gaps". An inexpensive open source darkfield biosensor system using Raspberry Pi imaging permits label-free detection and counting of colonies or plaques within 4-8 hours in a linear, liquid matrix within ∼200 µm inner diameter microcapillaries. We obtained full quantitative agreement between 1D microfluidic colony counting in dipsticks versus conventional 2D solid agar Petri dish plates for S. aureus and E. coli, and for T2 phage and phage K, but up to 6 times faster. Time-lapse darkfield imaging permitted detailed kinetic analysis of colony growth in the microcapillaries, providing new insight into microfluidic microbiology and colony growth, not possible with Petri dishes. Surprisingly, whilst E. coli colonies appeared earlier, subsequent colony expansion was faster along the microcapillaries for S. aureus. This may be explained by the microenvironment offered for 1D colony growth within microcapillaries, linked to a mass balance between nutrient (glucose) diffusion and bacterial growth kinetics. Counting individual colonies in liquid medium was not possible for motile strains that spread rapidly along the capillary, however inclusion of soft agar inhibited spreading, making this new simple dip-and-test counting method applicable to both motile and non-motile bacteria. Label-free dipstick colony and plaque counting has potential for many analytical microbial tasks, and the innovation of 1D colony counting has relevance to other microfluidic microbiology.

The impact of severe COVID-19 on health-related quality of life and disability: an early follow-up perspective. / Impacto da COVID-19 grave na qualidade de vida relacionada com a saúde e a incapacidade: uma perspectiva de follow-up a curto-prazo.

OBJECTIVE: To assess early postdischarge health-related quality of life and disability of all survivors of critical COVID-19 admitted for more than 24 hours to na intensive care unit.. METHODS: Study carried out at the Intensive Care Medicine Department of Centro Hospitalar Universitário São João from 8th October 2020 to 16th February 2021. Approximately 1 month after hospital discharge, an intensive care-trained nurse performed a telephone consultation with 99 survivors already at home applying the EuroQol Five-Dimensional Five-Level questionnaire and the 12-item World Health Organization Disability Assessment Schedule 2.0. RESULTS: The mean age of the population studied was 63 ± 12 years, and 32.5% were submitted to invasive mechanical ventilation. Their mean Simplified Acute Physiologic Score was 35 ± 14, and the Charlson Comorbidity Index was 3 ± 2. Intensive care medicine and hospital lengths of stay were 13 ± 22 and 22 ± 25 days, respectively. The mean EuroQol Visual Analog Scale was 65% (± 21), and only 35.3% had no or slight problems performing their usual activities, most having some degree of pain/discomfort and anxiety/depression. The 12-item World Health Organization Disability Assessment Schedule 2.0 showed marked impairments in terms of reassuring usual work or community activities and mobility. The use of both tools suggested that their health status was worse than their perception of it. CONCLUSION: This early identification of sequelae may help define flows and priorities for rehabilitation and reinsertion after critical COVID-19.

OBJETIVO: Avaliar a qualidade de vida relacionada com a saúde e a incapacidade no primeiro mês após a alta para domicílio de todos os sobreviventes de COVID-19 grave internados por mais de 24 horas no Serviço de Medicina Intensiva. METÓDOS: Estudo realizado no Serviço de Medicina Intensiva do Centro Hospitalar Universitário São João, entre 8 de outubro de 2020 e 16 de fevereiro de 2021. Aproximadamente 1 mês após a alta para domicílio, uma enfermeira com experiência em medicina intensiva realizou uma consulta telefônica a 99 sobreviventes, aplicando os questionários EuroQol Five-Dimensional Five-Level e World Health Disability Assessment Schedule 2.0 - 12 itens. RESULTADOS: A média de idade da população estudada foi de 63 ± 12 anos, e 32,5% foram submetidos à ventilação mecânica invasiva. O Simplified Acute Physiology Score médio foi de 35 ± 14, e o Índice de Comorbilidades de Charlson foi de 3 ± 2. O tempo de internamento em medicina intensiva e no hospital foi de 13 ± 22 e 22 ± 25 dias, respectivamente. A média da Escala Visual Analógica da EuroQol foi de 65% (± 21), sendo que apenas 35,3% dos sobreviventes não apresentaram ou tiveram problemas ligeiros para realizar suas atividades habituais, a maioria com algum grau de dor/desconforto e ansiedade/depressão. O World Health Disability Assessment Schedule 2.0 - 12 itens, mostrou incapacidade marcada em retomar o trabalho habitual ou atividades comunitárias e na mobilidade. O uso de ambas as ferramentas sugeriu que o estado de saúde dos sobreviventes seria pior do que a sua percepção. CONCLUSÃO: A identificação precoce de sequelas pode ajudar a definir fluxos e prioridades para a reabilitação e reinserção após a COVID-19 grave.

Prognostic factors and management of colorectal gastrointestinal stromal tumors

Introduction: The gastrointestinal stromal tumor (GIST) is the most common mesenchymal neoplasm of the gastrointestinal tract. Even though it can be found in any location of the digestive tract, the colorectal GIST is rare. With this study, we aim to review the current knowledge regarding the prognosis and management of colorectal GIST. Methods: A literature search was conducted in PubMed, and 717 articles were collected. After analyzing these studies, 60 articles were selected to use in this review. Results: The mitotic index, as well as tumor size and location were identified as good discriminators of prognosis in various studies. Surgery remains the only curative therapy for potentially resectable tumors. However, even after surgical resection, some patients develop disease recurrence and metastasis, especially those with highrisk tumors. Therefore, surgical resection alone might be inadequate for the management of all colorectal GISTs. The discovery of GIST's molecular pathway led to a shift in its therapy, insofar as tyrosine kinase inhibitors became part of the treatment schemes for this tumor, revolutionizing the treatment's outcome and prognosis. Discussion/Conclusion: The controversy concerning colorectal GIST prognosis and treatment can be, in part, attributed to the limited number of studies in the literature. In this review, we gathered the most recent knowledge about the prognosis and management of GIST in this rare location and propose two algorithms for its approach. Lastly, we highlight the importance of an individualized approach in the setting of a multidisciplinary team. (AU)

Impacto da COVID-19 grave na qualidade de vida relacionada com a saúde e a incapacidade: uma perspectiva de follow-up a curto-prazo / The impact of severe COVID-19 on health-related quality of life and disability: an early follow-up perspective

RESUMO Objetivo: Avaliar a qualidade de vida relacionada com a saúde e a incapacidade no primeiro mês após a alta para domicílio de todos os sobreviventes de COVID-19 grave internados por mais de 24 horas no Serviço de Medicina Intensiva. Metódos: Estudo realizado no Serviço de Medicina Intensiva do Centro Hospitalar Universitário São João, entre 8 de outubro de 2020 e 16 de fevereiro de 2021. Aproximadamente 1 mês após a alta para domicílio, uma enfermeira com experiência em medicina intensiva realizou uma consulta telefônica a 99 sobreviventes, aplicando os questionários EuroQol Five-Dimensional Five-Level e World Health Disability Assessment Schedule 2.0 - 12 itens. Resultados: A média de idade da população estudada foi de 63 ± 12 anos, e 32,5% foram submetidos à ventilação mecânica invasiva. O Simplified Acute Physiology Score médio foi de 35 ± 14, e o Índice de Comorbilidades de Charlson foi de 3 ± 2. O tempo de internamento em medicina intensiva e no hospital foi de 13 ± 22 e 22 ± 25 dias, respectivamente. A média da Escala Visual Analógica da EuroQol foi de 65% (± 21), sendo que apenas 35,3% dos sobreviventes não apresentaram ou tiveram problemas ligeiros para realizar suas atividades habituais, a maioria com algum grau de dor/desconforto e ansiedade/depressão. O World Health Disability Assessment Schedule 2.0 - 12 itens, mostrou incapacidade marcada em retomar o trabalho habitual ou atividades comunitárias e na mobilidade. O uso de ambas as ferramentas sugeriu que o estado de saúde dos sobreviventes seria pior do que a sua percepção. Conclusão: A identificação precoce de sequelas pode ajudar a definir fluxos e prioridades para a reabilitação e reinserção após a COVID-19 grave.

ABSTRACT Objective: To assess early postdischarge health-related quality of life and disability of all survivors of critical COVID-19 admitted for more than 24 hours to na intensive care unit.. Methods: Study carried out at the Intensive Care Medicine Department of Centro Hospitalar Universitário São João from 8th October 2020 to 16th February 2021. Approximately 1 month after hospital discharge, an intensive care-trained nurse performed a telephone consultation with 99 survivors already at home applying the EuroQol Five-Dimensional Five-Level questionnaire and the 12-item World Health Organization Disability Assessment Schedule 2.0. Results: The mean age of the population studied was 63 ± 12 years, and 32.5% were submitted to invasive mechanical ventilation. Their mean Simplified Acute Physiologic Score was 35 ± 14, and the Charlson Comorbidity Index was 3 ± 2. Intensive care medicine and hospital lengths of stay were 13 ± 22 and 22 ± 25 days, respectively. The mean EuroQol Visual Analog Scale was 65% (± 21), and only 35.3% had no or slight problems performing their usual activities, most having some degree of pain/discomfort and anxiety/depression. The 12-item World Health Organization Disability Assessment Schedule 2.0 showed marked impairments in terms of reassuring usual work or community activities and mobility. The use of both tools suggested that their health status was worse than their perception of it. Conclusion: This early identification of sequelae may help define flows and priorities for rehabilitation and reinsertion after critical COVID-19.

Gravity-Driven Microfluidic Siphons: Fluidic Characterization and Application to Quantitative Immunoassays.

A range of biosensing techniques including immunoassays are routinely used for quantitation of analytes in biological samples and available in a range of formats, from centralized lab testing (e.g., microplate enzyme-linked immunosorbent assay (ELISA)) to automated point-of-care (POC) and lateral flow immunochromatographic tests. High analytical performance is intrinsically linked to the use of a sequence of reagent and washing steps, yet this is extremely challenging to deliver at the POC without a high level of fluidic control involving, e.g., automation, fluidic pumping, or manual fluid handling/pipetting. Here we introduce a microfluidic siphon concept that conceptualizes a multistep ″dipstick″ for quantitative, enzymatically amplified immunoassays using a strip of microporous or microbored material. We demonstrated that gravity-driven siphon flow can be realized in single-bore glass capillaries, a multibored microcapillary film, and a glass fiber porous membrane. In contrast to other POC devices proposed to date, the operation of the siphon is only dependent on the hydrostatic liquid pressure (gravity) and not capillary forces, and the unique stepwise approach to the delivery of the sample and immunoassay reagents results in zero dead volume in the device, no reagent overlap or carryover, and full start/stop fluid control. We demonstrated applications of a 10-bore microfluidic siphon as a portable ELISA system without compromised quantitative capabilities in two global diagnostic applications: (1) a four-plex sandwich ELISA for rapid smartphone dengue serotype identification by serotype-specific dengue virus NS1 antigen detection, relevant for acute dengue fever diagnosis, and (2) quantitation of anti-SARS-CoV-2 IgG and IgM titers in spiked serum samples. Diagnostic siphons provide the opportunity for high-performance immunoassay testing outside sophisticated laboratories, meeting the rapidly changing global clinical and public health needs.

Antibody Surface Coverage Drives Matrix Interference in Microfluidic Capillary Immunoassays.

The performance of biosensors is often optimized in buffers, which brings inconsistencies during applications with biological samples. Current strategies for minimizing sample (matrix) interference are complex to automate and miniaturize, involving, e.g., sample dilution or recovery of serum/plasma. This study shows the first systematic analysis using hundreds of actual microfluidic immunoassay fluoropolymer strips to understand matrix interference in microflow systems. As many interfering factors are assay-specific, we have explored matrix interference for a range of enzymatic immunoassays, including a direct mIgG/anti-mIgG, a sandwich cancer biomarker PSA, and a sandwich inflammatory cytokine IL-1ß. Serum matrix interference was significantly affected by capillary antibody surface coverage, suggesting for the first time that the main cause of the serum matrix effect is low-affinity serum components (e.g., autoantibodies) competing with high-affinity antigens for the immobilized antibody. Additional experiments carried out with different capillary diameters confirmed the importance of antibody surface coverage in managing matrix interference. Building on these findings, we propose a novel analytical approach where antibody surface coverage and sample incubation times are key for eliminating and/or minimizing serum matrix interference, consisting in bioassay optimization carried out in serum instead of buffer, without compromising the performance of the bioassay or adding extra cost or steps. This will help establishing a new route toward faster development of modern point-of-care tests and effective biosensor development.

Microfluidic smartphone quantitation of Escherichia coli in synthetic urine.

In spite of the clinical need, there is a major gap in rapid diagnostics for identification and quantitation of E. coli and other pathogens, also regarded as the biggest bottleneck in the fight against the spread of antimicrobial resistant bacterial strains. This study reports for the first time an optical, smartphone-based microfluidic fluorescence sandwich immunoassay capable of quantifying E. coli in buffer and synthetic urine in less than 25 min without sample preparation nor concentration. A limit of detection (LoD) up to 240 CFU/mL, comensurate with cut-off for UTIs (103-105 CFUs/mL) was achieved. Replicas of full response curves performed with 100-107 CFUs/mL of E. coli K12 in synthetic urine yielded recovery values in the range 80-120%, assay reproducibility below 30% and precision below 20%, therefore similar to high-performance automated immunoassays. The unrivalled LoD was mainly linked to the 'open fluidics' nature of the 10-bore microfluidic strips used that enabled passing a large volume of sample through the microcapillaries coated with capture antibody. The new smartphone based test has the potential of being as a rapid, point-of-care test for rule-in of E. coli infections that are responsible for around 80% of UTIs, helping to stop the over-prescription of antibiotics and the monitoring of patients with other symptomatic communicable diseases caused by E. coli at global scale.

Transparent, Hydrophobic Fluorinated Ethylene Propylene Offers Rapid, Robust, and Irreversible Passive Adsorption of Diagnostic Antibodies for Sensitive Optical Biosensing.

Current literature data is scarce and somehow contradictory in respect to the suitability of "nonstick" fluoropolymer surfaces for immobilization of biomolecules. We have previously shown empirically that transparent Teflon fluorinated ethylene propylene (FEP) offers rapid and sensitive optical biosensing of clinically relevant biomarkers. This study shows for the first time a comprehensive experimental analysis of passive adsorption of diagnostic IgG antibodies on actual Teflon FEP microfluidic strips. Full equilibrium isotherms and kinetics for passive adsorption were studied and modeled employing a protein titration method using hundreds of multibore microfluidic strips for a range of temperatures, pH, ionic strengths, and inner diameters, using both polyclonal and monoclonal antibody systems. Results were benchmarked against other plastic hydrophobic and glass hydrophilic capillary surfaces. For the first time, it was shown quantitatively that the hydrophobicity of fluoropolymer surfaces encourages the passive adsorption of diagnostic antibodies for biosensing and is insensitive to the temperature of incubation and to ionic buffer strength. The mass of captured antigen increased with increasing antibody surface coverage up to ∼400 ng/cm2, with an optimal adsorbed antibody activity for 45-69% of full monolayer coverage, matching results of other biosensing surfaces. The equilibrium was reached fast, within 5-10 min, and surprisingly both the kinetics and equilibrium of antibody adsorption were dependent on the inner diameter of microcapillaries. This is a novel and relevant result that will generally impact on the design of miniaturized microfluidic biosensing devices. The antibody surface densities obtained with hydrophobic plastic surfaces were 2- to 4-fold lower than for a hydrophilic, glass surface, however the former presented a monolayered adsorption with a higher level of irreversibility, as shown by the adsorption and desorption rates around 1 order of magnitude smaller than for glass, which is highly desirable for biosensing with surface-coated biomolecules.

Immunocapture of Escherichia coli in a fluoropolymer microcapillary array.

This study presents novel experimental insights into the direct quantitation and immunocapture of bacteria cells in a fluoropolymer microcapillary array, using Escherichia coli as work model, a pathogen responsible for around 80% of urinary tract infections (UTIs). In spite of the current clinical demand for sensitive tests for rapid identification and quantitation of pathogens in human samples, portable diagnostic tests developed to date lack the specificity, limit of detection and speed for effective implementation in bacteria detection at point-of-care. The 'open microfluidic' approach presented in this work directly addresses those challenges. We report for the first time evidence of immunocapture of bacteria using polyclonal antibodies immobilized on the inner surface of an inexpensive 10-bore, 200 µm internal diameter FEP-Teflon® MicroCapillary Film, with a limit of detection (LoD) of at just 1 colony forming unit (CFU). In capillaries coated with less than a full monolayer of capture antibody, we observed a first order equilibrium, with bacteria captured (in CFUs/ml) linearly proportional to the CFU/ml in the incubated sample. We captured up to 100% of E. coli cells, with clear evidence of immunospecificity as demonstrated by testing with a different bacteria specie (in this case Bacillus subtillis). We noticed gravity settling of bacteria within the capillaries created a gradient of concentration which on the overall enhanced the capturing of cells up to 6 orders of magnitude beyond the theoretic full monolayer (∼4.5 × 104 CFUs/ml), with washings having an unnoticeable effect. Our data particularly highlights quantitatively the relevance of interrogation volume in respect to the miniaturisation of bacteria quantitation, which cannot be solved with the most sophisticated imaging equipment. A further set of continuous flow experiments at a flow rate of just ∼1 µl/min (corresponding to a wall shear rate of ∼101 s-1 and superficial flow velocity ∼53 µm/s) showed a degree of flow focusing, yet the mobility, antibody affinity capturing and gravity settling of bacteria cells enabled successful capturing in the microcapillaries. These results will inform the future development of effective microfluidic approaches for rapid point-of-care quantitation of bacterial pathogens and in particular rule-in of E. coli in UTIs.

Removal of antiretroviral drugs stavudine and zidovudine in water under UV254 and UV254/H2O2 processes: Quantum yields, kinetics and ecotoxicology assessment.

The concentration of antiretroviral drugs in wastewater treatment plants (WWTP) effluents and surface waters of many countries has increased significantly due to their widespread use for HIV treatment. In this study, the removal of stavudine and zidovudine under UV254 photolysis or UV254/H2O2 was investigated in a microcapillary film (MCF) photoreactor, using minimal water samples quantities. The UV254 quantum yield of zidovudine, (2.357 ±â€¯0.0589)·10-2 mol ein-1 (pH 4.0-8.0), was 28-fold higher that the yield of stavudine (8.34 ±â€¯0.334)·10-4 mol ein-1 (pH 6.0-8.0). The second-order rate constant kOH,iof reaction of hydroxyl radical with the antiretrovirals (UV254/H2O2 process) were determined by kinetics modeling: (9.98 ±â€¯0.68)·108 M-1 s-1 (pH 4.0-8.0) for zidovudine and (2.03 ±â€¯0.18)·109 M-1 s-1 (pH 6.0-8.0) for stavudine. A battery of ecotoxicological tests (i.e. inhibition growth, bioluminescence, mutagenic and genotoxic activity) using bacteria (Aliivibrio fischeri, Salmonella typhimurium), crustacean (Daphnia magna) and algae (Raphidocelis subcapitata) revealed a marked influence of the UV dose on the ecotoxicological activity. The UV254/H2O2 treatment process reduced the ecotoxicological risk associated to direct photolysis of the antiretrovirals aqueous solutions, but required significantly higher UV254 doses (≥2000 mJ cm-2) in comparison to common water UV disinfection processes.

Towards One-Step Quantitation of Prostate-Specific Antigen (PSA) in Microfluidic Devices: Feasibility of Optical Detection with Nanoparticle Labels.

Rapid and quantitative prostate-specific antigen (PSA) biomarker detection would be beneficial to cancer diagnostics, improving early detection and therefore increasing chances of survival. Nanoparticle-based detection is routinely used in one-step nitrocellulose-based lateral flow (LF) immunoassays; however, it is well established within the scientific diagnostic community that LF technology lacks sensitivity for measuring biomarkers, such as prostate-specific antigen (PSA). A trend in point-of-care (POC) protein biomarker quantitation is the miniaturization of immunoassays in microfluidic devices. This work aimed at testing the feasibility of carbon and gold nanoparticles as immunoassay labels for PSA detection with cost-effective optical detection in a novel microfluidic POC platform called microcapillary film (MCF), consisting of a parallel array of fluoropolymer microcapillaries with 200-µm internal diameter. With neutravidin-coated carbon, nanoparticles were able to quantify an immobilized biotinylated monoclonal antibody (coating solution from 10 to 40 µg/ml) and PSA was successfully quantified in a sandwich assay using silver-enhanced gold nanoparticles and a flatbed scanner; yet, the dynamic range was limited to 10-100 ng/ml. Although direct optical detection of PSA without enzymatic amplification or fluorophores is possible and technically appealing for the simplified fluidics and signal scanning setups involved, ultimately, the binding of a thin layer of nanoparticles onto the wall of transparent microcapillaries is not sufficient to cause a significant drop on the optical colorimetric signal. Future studies will explore the use of fluorescence nanoparticles.

Spontaneous pseudomeningocele associated with lumbar spondylolisthesis: A case report and review of the literature.

BACKGROUND: Pseudomeningocele, an extradural collection of cerebrospinal fluid (CSF), has only been rarely reported to occur spontaneously in conjunction with isthmic spondylolisthesis (with lysis) in the lumbar spine. CASE DESCRIPTION: A 68-year-old male presented with low back pain and neurogenic claudication of several years duration without any history of trauma, epidural spine injections, or spine surgery. Lumbosacral magnetic resonance imaging (MRI) revealed a grade-I L4-L5 isthmic spondylolisthesis with spinal canal narrowing and a posterior paravertebral collection consistent with CSF. The patient underwent a spinal decompression consisting of a complete L4 and partial L5 laminectomy, a bilateral L4-L5 instrumented fusion (due to the lysis defect), and closure of the CFS fistula. The histology analysis was compatible with a pseudomeningocele. CONCLUSION: Lumbar isthmic spondylolisthesis may lead to changes in the elastic properties of the underlying dura mater. Rarely, this may lead to meningeal tears and formation of a pseudomeningocele. Historically, one must always check for a prior epidural injection that could have resulted in this complication. Additionally, as most likely the case here, the lysis defect was responsible for the foraminal dural laceration resulting in the pseudomeningocele.

Photodegradation and ecotoxicology of acyclovir in water under UV254 and UV254/H2O2 processes.

The photochemical and ecotoxicological fate of acyclovir (ACY) through UV254 direct photolysis and in the presence of hydroxyl radicals (UV254/H2O2 process) were investigated in a microcapillary film (MCF) array photoreactor, which provided ultrarapid and accurate photochemical reaction kinetics. The UVC phototransformation of ACY was found to be unaffected by pH in the range from 4.5 to 8.0 and resembled an apparent autocatalytic reaction. The proposed mechanism included the formation of a photochemical intermediate (ϕACY = (1.62 ± 0.07)·10-3 mol ein-1) that further reacted with ACY to form by-products (k' = (5.64 ± 0.03)·10-3 M-1 s-1). The photolysis of ACY in the presence of hydrogen peroxide accelerated the removal of ACY as a result of formation of hydroxyl radicals. The kinetic constant for the reaction of OH radicals with ACY (kOH/ACY) determined with the kinetic modeling method was (1.23 ± 0.07)·109 M-1 s-1 and with the competition kinetics method was (2.30 ± 0.11)·109 M-1 s-1 with competition kinetics. The acute and chronic effects of the treated aqueous mixtures on different living organisms (Vibrio fischeri, Raphidocelis subcapitata, D. magna) revealed significantly lower toxicity for the samples treated with UV254/H2O2 in comparison to those collected during UV254 treatment. This result suggests that the addition of moderate quantity of hydrogen peroxide (30-150 mg L-1) might be a useful strategy to reduce the ecotoxicity of UV254 based sanitary engineered systems for water reclamation.

A critical insight into the development pipeline of microfluidic immunoassay devices for the sensitive quantitation of protein biomarkers at the point of care.

The latest clinical procedures for the timely and cost-effective diagnosis of chronic and acute clinical conditions, such as cardiovascular diseases, cancer, chronic respiratory diseases, diabetes or sepsis (i.e. the biggest causes of death worldwide), involve the quantitation of specific protein biomarkers released into the blood stream or other physiological fluids (e.g. urine or saliva). The clinical thresholds are usually in the femtomolar to picolomar range, and consequently the measurement of these protein biomarkers heavily relies on highly sophisticated, bulky and automated equipment in centralised pathology laboratories. The first microfluidic devices capable of measuring protein biomarkers in miniaturised immunoassays were presented nearly two decades ago and promised to revolutionise point-of-care (POC) testing by offering unmatched sensitivity and automation in a compact POC format; however, the development and adoption of microfluidic protein biomarker tests has fallen behind expectations. This review presents a detailed critical overview into the pipeline of microfluidic devices developed in the period 2005-2016 capable of measuring protein biomarkers from the pM to fM range in formats compatible with POC testing, with a particular focus on the use of affordable microfluidic materials and compact low-cost signal interrogation. The integration of these two important features (essential unique selling points for the successful microfluidic diagnostic products) has been missed in previous review articles and explain the poor adoption of microfluidic technologies in this field. Most current miniaturised devices compromise either on the affordability, compactness and/or performance of the test, making current tests unsuitable for the POC measurement of protein biomarkers. Seven core technical areas, including (i) the selected strategy for antibody immobilisation, (ii) the surface area and surface-area-to-volume ratio, (iii) surface passivation, (iv) the biological matrix interference, (v) fluid control, (vi) the signal detection modes and (vii) the affordability of the manufacturing process and detection system, were identified as the key to the effective development of a sensitive and affordable microfluidic protein biomarker POC test.

Covalent immobilisation of antibodies in Teflon-FEP microfluidic devices for the sensitive quantification of clinically relevant protein biomarkers.

This study reports for the first time the sensitive colorimetric and fluorescence detection of clinically relevant protein biomarkers by sandwich immunoassays using the covalent immobilisation of antibodies onto the fluoropolymer surface inside Teflon®-FEP microfluidic devices. Teflon®-FEP has outstanding optical transparency ideal for high-sensitivity colorimetric and fluorescence bioassays, however this thermoplastic is regarded as chemically inert and very hydrophobic. Covalent immobilisation can offer benefits over passive adsorption to plastic surfaces by allowing better control over antibody density, orientation and analyte binding capacity, and so we tested a range of different and novel covalent immobilisation strategies. We first functionalised the inner surface of a 10-bore, 200 µm internal diameter FEP microcapillary film with high-molecular weight polyvinyl alcohol (PVOH) without changing the outstanding optical transparency of the device delivered by the matched refractive index of FEP and water. Glutaraldehyde immobilisation was compared with the use of photoactivated linkers and NHS-ester crosslinkers for covalently immobilising capture antibodies onto PVOH. Three clinically relevant sandwich ELISAs were tested against the cytokine IL-1ß, the myocardial infarct marker cardiac troponin I (cTnI), and the chronic heart failure marker brain natriuretic peptide (BNP). Overall, glutaraldehyde immobilisation was effective for BNP assays, but yielded unacceptable background for IL-1ß and cTnI assays caused by direct binding of the biotinylated detection antibody to the modified PVOH surface. We found NHS-ester groups reacted with APTES-treated PVOH coated fluoropolymers. This facilitated a novel method for capture antibody immobilisation onto fluoropolymer devices using a bifunctional NHS-maleimide crosslinker. The density of covalently immobilised capture antibodies achieved using PVOH/APTES/NHS/maleimide approached levels seen with passive adsorption, and sensitive and quantitative assay performance was achieved using this method. Overall, the PVOH coating provided an excellent surface for controlled covalent antibody immobilisation onto Teflon®-FEP for performing high-sensitivity immunoassays.

Lab on a stick: multi-analyte cellular assays in a microfluidic dipstick.

A new microfluidic concept for multi-analyte testing in a dipstick format is presented, termed "Lab-on-a-Stick", that combines the simplicity of dipstick tests with the high performance of microfluidic devices. Lab-on-a-stick tests are ideally suited to analysis of particulate samples such as mammalian or bacterial cells, and capable of performing multiple different parallel microfluidic assays when dipped into a single sample with results recorded optically. The utility of this new diagnostics format was demonstrated by performing three types of multiplex cellular assays that are challenging to perform in conventional dipsticks: 1) instantaneous ABO blood typing; 2) microbial identification; and 3) antibiotic minimum inhibitory (MIC) concentration measurement. A pressure balance model closely predicted the superficial flow velocities in individual capillaries, that were overestimated by up to one order of magnitude by the Lucas-Washburn equation conventionally used for wicking in cylindrical pores. Lab-on-a-stick provides a cost-effective, simple, portable and flexible multiplex platform for a range of assays, and will deliver a new generation of advanced yet affordable point-of-care tests for global diagnostics.

Investigation on the removal of the major cocaine metabolite (benzoylecgonine) in water matrices by UV254/H2O2 process by using a flow microcapillary film array photoreactor as an efficient experimental tool.

A microcapillary film reactor (MCF) was adopted to evaluate and compare the removal efficiency of benzoylecgonine (BE), an emerging micropollutant deriving from illicit drug abuse (cocaine), in different aqueous matrices: milliQ water, synthetic and real wastewater and surface water. The removal processes investigated were the direct photolysis with UV radiation at 254 nm, and the advanced oxidation process (AOP) with the same UV radiation and hydrogen peroxide. As a result of the microfluidics approach developed through an innovative experimental apparatus, full conversion of BE was reached within a few seconds or minutes of residence time in the MCF depending on the process conditions adopted. The radiation dose was estimated to be approximately 5.5 J cm(-2). The innovative MCF reactor was found to be an effective tool for photochemical studies, especially when using highly priced, uncommon, or regulated substances. The removal efficiency was affected by the nature of the aqueous matrix, due to the presence of different xenobiotics and natural compounds that act primarily as HO(•) radical scavengers and secondly as inner UV254 filters. Moreover, nano-liquid chromatography (LC)-high resolution-mass spectrometry analysis was utilized to identify the main reaction transformation products, showing the formation of hydroxylated aromatics during the photochemical treatment.