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Considering the need for a more time and cost-effective method for lamotrigine (LTG) detection in clinics we developed a fast and robust label-free assay based on surface-enhanced Raman scattering (SERS) for LTG quantification from human serum. The optimization and application of the developed assay is presented showing the: (i) exploration of different methods for LTG separation from human serum; (ii) implementation of a molecular adsorption step on an ordered Au nanopillar SERS substrate; (iii) adaptation of a fast scanning of the SERS substrate, performed with a custom-built compact Raman spectrometer; and (iv) development of LTG quantification methods with univariate and multivariate spectral data analysis. Our results showed, for the first time, the SERS-based characterization of LTG and its label-free identification in human serum. We found that combining a miniaturized solid phase extraction, as sample pre-treatment with the SERS assay, and using a multivariate model is an optimal strategy for LTG quantification in human serum in a linear range from 9.5 to 75 µM, with LoD and LoQ of 3.2 µM and 9.5 µM, respectively, covering the suggested clinical therapeutic window. We also showed that the developed assay allowed for quantifying LTG from human serum in the presence of other drugs, thereby demonstrating the robustness of label-free SERS. The sensing approach and instrumentation can be further automated and integrated in devices that can advance the drug monitoring in real clinical settings.
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Anticonvulsivantes , Espectrometría Raman , Humanos , Lamotrigina , Espectrometría Raman/métodos , Análisis de DatosRESUMEN
For patients with ulcerative colitis (UC), administration of the probiotic E. coli Nissle (EcN) holds promise for alleviation of disease symptoms. The mechanisms are unclear, but it has been hypothesised that a capacity of the probiotic to outcompete potentially detrimental UC-associated E. coli strains plays an important role. However, this could previously not be confirmed in a mouse model of competition between EcN and two UC-associated strains, as reported by Petersen et al. 2011. In the present study, we re-evaluated the idea, hypothesising that delivery of EcN by a micro device dosing system (microcontainers), designed for delivery into the intestinal mucus, could support colonisation and confer a competition advantage compared to classical oral dosing. Six groups of mice were pre-colonised with one of two UC-associated E. coli strains followed by oral delivery of EcN, either in capsules containing microcontainers with freeze-dried EcN powder, capsules containing freeze-dried EcN powder, or as a fresh sucrose suspension. Co-colonisation between the probiotic and the disease-associated strains was observed regardless of dosing method, and no competition advantages linked to microcontainer delivery were identified within this setup. Other approaches are thus needed if the competitive capacity of EcN in the gut should be improved.
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Colitis Ulcerosa , Probióticos , Humanos , Ratones , Animales , Colitis Ulcerosa/inducido químicamente , Escherichia coli , PolvosRESUMEN
We present an unconventional approach to a common Lab-on-a-Disc (LoD) that combines a quadcopter propulsion system, a miniaturized 2.4 GHz Wi-Fi spy camera, 9.74 Watt Qi wireless power, and an Arduino into an open-source, miniaturized All-in-one powered lab-on-disc platform (APELLA). The quadcopter propulsion generates thrust to rotate (from 0.1 to 24.5 Hz) or shake the LoD device, while the spy camera enables a real-time (30 frames per second) and high definition (1280 × 720 pixels) visualization of microfluidic channels without requiring a bulky and heavy stroboscopic imaging setup. A mobile device can communicate with an Arduino microcontroller inside the APELLA through a Bluetooth interface for closed loop and sequential frequency control. In a proof-of-concept study, the APELLA achieved comparable mixing efficiency to a traditional spin stand and can capture rapid microfluidic events at low rotational frequencies (<5Hz). The APELLA is low-cost (c.a. 100 Euro), compact (15.6 × 15.6 × 10 cm3), lightweight (0.59 kg), portable (powered by a 5 V USB power bank), and energy efficient (uses < 6% power of the conventional system), making it ideal for field deployment, education, resource-limited labs.
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In vitro, cell-based assays are essential in diagnostics and drug development. There are ongoing efforts to establish new technologies that enable real-time detection of cell-drug interaction during culture under flow conditions. Our compact (10 × 10 × 8.5 cm) cell culture and microscope on disc (CMoD) platform aims to decrease the application barriers of existing lab-on-a-chip (LoC) approaches. For the first time in a centrifugal device, (i) cells were cultured for up to six days while a spindle motor facilitated culture medium perfusion, and (ii) an onboard microscope enabled live bright-field imaging of cells while the data wirelessly transmitted to a computer. The quantification of cells from the acquired images was done using artificial intelligence (AI) software. After optimization, the obtained cell viability data from the AI-based image analysis proved to correlate well with data collected from commonly used image analysis software. The CMoD was also suitable for conducting a proof-of-concept toxicity assay with HeLa cells under continuous flow. The half-maximal inhibitory time (IT50) for various concentrations of doxorubicin (DOX) in the case of HeLa cells in flow, was shown to be lower than the IT50 obtained from a static cytotoxicity assay, indicating a faster onset of cell death in flow. The CMoD proved to be easy to handle, enabled cell culture and monitoring without assistance, and is a promising tool for examining the dynamic processes of cells in real-time assays.
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Inteligencia Artificial , Procesamiento de Imagen Asistido por Computador , Humanos , Células HeLa , Microscopía , PerfusiónRESUMEN
During the past decades, microdevices have been evaluated as a means to overcome challenges within oral drug delivery, thus improving bioavailability. Fabrication of microdevices is often limited to planar or simple 3D designs. Therefore, this work explores how microscale stereolithography 3D printing can be used to fabricate radiopaque microcontainers with enhanced mucoadhesive geometries, which can enhance bioavailability by increasing gastrointestinal retention. Ex vivo force measurements suggest increased mucoadhesion of microcontainers with adhering features, such as pillars and arrows, compared to a neutral design. In vivo studies, utilizing planar X-ray imaging, show the time-dependent gastrointestinal location of microcontainers, whereas computed tomography scanning and cryogenic scanning electron microscopy reveal information about their spatial dynamics and mucosal interactions, respectively. For the first time, the effect of 3D microdevice modifications on gastrointestinal retention is traced in vivo, and the applied methods provide a much-needed approach for investigating the impact of device design on gastrointestinal retention.
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Sistemas de Liberación de Medicamentos , Tomografía Computarizada por Rayos X , Sistemas de Liberación de Medicamentos/métodos , Disponibilidad Biológica , Microscopía Electrónica de Rastreo , Impresión TridimensionalRESUMEN
The biggest challenge in oral delivery of anti-inflammatory drugs such as 5-aminosalicylic acid (5-ASA) is to (i) prevent rapid absorption in the small intestine and (ii) achieve localized release at the site of inflammation in the lower gut, i.e., the colon. Here, we present an advanced biopolymeric coating comprising of tannic-acid-functionalized zein protein to provide a sustained, colon-targeted release profile for 5-ASA and enhance the mucoadhesion of the dosage form via a mussel-inspired mechanism. To enable localized delivery and provide high local concentration, 5-ASA is loaded into the microfabricated drug carriers (microcontainers) and sealed with the developed coating. The functionality and drug release profile of the coating are characterized and optimized in vitro, showing great tunability, scalability, and stability toward proteases. Further, ex vivo experiments demonstrate that the tannic acid functionalization can significantly enhance the mucoadhesion of the coating, which is followed up by in vivo investigations on the intestinal retention, and pharmacokinetic evaluation of the 5-ASA delivery system. Results indicate that the developed coating can provide prolonged colonic delivery of 5-ASA. Therefore, the here-developed biodegradable coating can be an eco-friendly substitute to the state-of-the-art commercial counterparts for targeted delivery of 5-ASA and other small molecule drugs.
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Therapeutic drug monitoring (TDM) is an essential clinical practice for optimizing drug dosing, thereby preventing adverse effects of drugs with a narrow therapeutic window, slow clearance, or high interperson pharmacokinetic variability. Monitoring methotrexate (MTX) during high-dose MTX (HD-MTX) therapy is necessary to avoid potentially fatal side effects caused by delayed elimination. Despite the efficacy of HD-MTX treatment, its clinical application in resource-limited settings is constrained due to the relatively high cost and time of analysis with conventional analysis methods. In this work, we developed (i) an electrochemically assisted surface-enhanced Raman spectroscopy (SERS) method for detecting MTX in human serum at a clinically relevant concentration range and (ii) a benchtop, Raman detection system with an integrated potentiostat, software, and data analysis unit that enables mapping of small areas of SERS substrates and quantitative SERS-based analysis. In the assay, by promoting electrostatic attraction between gold-coated nanopillar SERS substrates and MTX molecules in aqueous samples, a detection limit of 0.13 µM with a linear range of 0.43-2 µM was achieved in PBS. The implemented sample cleanup through gel filtration proved to be highly effective, resulting in a similar detection limit (0.55 µM) and linear range (1.81-5 µM) for both PBS and serum. The developed and optimized assay could also be used on the in-house built, Raman device. We showed that MTX detection can be carried out in less than 30 min with the Raman device, paving the way toward the TDM of MTX at the point-of-need and in resource-limited environments.
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Metotrexato , Espectrometría Raman , Bioensayo , Monitoreo de Drogas/métodos , Oro/química , Humanos , Espectrometría Raman/métodosRESUMEN
Oral antibiotic treatment is often applied in animal studies in order to allow establishment of an introduced antibiotic-resistant bacterium in the gut. Here, we compared the application of streptomycin dosed orally in microcontainers to dosage through drinking water. The selective effect on a resistant bacterial strain, as well as the effects on fecal, luminal, and mucosal microbiota composition, were investigated. Three groups of rats (n = 10 per group) were orally dosed with microcontainers daily for 3 days. One of these groups (STR-M) received streptomycin-loaded microcontainers designed for release in the distal ileum, while the other two groups (controls [CTR] and STR-W) received empty microcontainers. The STR-W group was additionally dosed with streptomycin through the drinking water. A streptomycin-resistant Escherichia coli strain was orally inoculated into all animals. Three days after inoculation, the resistant E. coli was found only in the cecum and colon of animals receiving streptomycin in microcontainers but in all intestinal compartments of animals receiving streptomycin in the drinking water. 16S rRNA amplicon sequencing revealed significant changes in the fecal microbiota of both groups of streptomycin-treated animals. Investigation of the inner colonic mucus layer by confocal laser scanning microscopy and laser capture microdissection revealed no significant effect of streptomycin treatment on the mucus-inhabiting microbiota or on E. coli encroachment into the inner mucus. Streptomycin-loaded microcontainers thus enhanced proliferation of an introduced streptomycin-resistant E. coli in the cecum and colon without affecting the small intestine environment. While improvements of the drug delivery system are needed to facilitate optimal local concentration and release of streptomycin, the application of microcontainers provides new prospects for antibiotic treatment. IMPORTANCE Delivery of antibiotics in microcontainer devices designed for release at specific sites of the gut represents a novel approach which might reduce the amount of antibiotic needed to obtain a local selective effect. We propose that the application of microcontainers may have the potential to open novel opportunities for antibiotic treatment of humans and animals with fewer side effects on nontarget bacterial populations. In the current study, we therefore elucidated the effects of streptomycin, delivered in microcontainers coated with pH-sensitive lids, on the selective effect on a resistant bacterium, as well as on the surrounding intestinal microbiota in rats.
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Agua Potable , Estreptomicina , Animales , Antibacterianos/farmacología , Antibacterianos/uso terapéutico , Bacterias/genética , Colon , Escherichia coli/genética , Humanos , Mucosa Intestinal/microbiología , ARN Ribosómico 16S , Ratas , Estreptomicina/farmacologíaRESUMEN
In vitro release and dissolution models are widely used in the development phases of oral drug delivery systems to measure how an active pharmaceutical ingredient (API) is released from a dosage form. However, additional requirements for these models arise when evaluating probiotic dosage forms since they are often sensitive to temperature and oxygen levels. As a solution to this, we propose a custom-designed anaerobic in vitro release setup, made mainly by 3D printing and laser cutting, to function together with state-of-the-art pharmaceutical dissolution equipment - in this case, a microDISS Profiler™. The in vitro release model makes it possible to study the release rate of oxygen-sensitive probiotics in simulated intestinal conditions, while ensuring their survival due to the anaerobic conditions. This has not been possible so far since the available in vitro dissolution models have not been compatible with anaerobic conditions. With two different case studies, the developed model combined with a microDISS Profiler™ has proven capable of measuring the release of a probiotic and a small-molecule API from microdevices for oral drug delivery. Further, the model facilitated the survival of anaerobic bacteria present in the release medium.
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Therapeutic drug monitoring (TDM) can improve clinical care when using drugs with pharmacokinetic variability and a narrow therapeutic window. Rapid, reliable, and easy-to-use detection methods are required in order to decrease the time of analysis and can also enable TDM in resource-limited settings or even at bedside. Monitoring methotrexate (MTX), an anticancer drug, is critical since it is needed to follow the drug clearance rate and decide how to administer the rescue drug, leucovorin (LV), in order to avoid toxicity and even death. We show that with the optimized nanopillar-assisted separation (NPAS) method using surface-enhanced Raman scattering, we were able to measure MTX in PBS and serum in the linear range of 5-150 µM and confirmed that MTX detection can be carried out even in the presence of LV. Additionally, when NPAS was combined with centrifugal filtration, a quantification limit of 2.1 µM for MTX in human serum sample was achieved. The developed detection method enables fast detection (10 min) and quantification of MTX from human serum (>90% accuracy). Furthermore, we show the potential of the developed method for TDM, when quantifying MTX from clinical samples, collected from patients who are undergoing high-dose MTX therapy.
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Monitoreo de Drogas , Metotrexato , Humanos , Leucovorina , Espectrometría RamanRESUMEN
Complex and miniaturized oral drug delivery systems are being developed rapidly for targeted, controlled drug release and improved bioavailability. Standard analytical techniques are widely used to characterize i) drug carrier and active pharmaceutical ingredients before loading into a delivery device (to ensure the solid form), and ii) the entire drug delivery system during the development process. However, in light of the complexity and the size of some of these systems, standard techniques as well as novel sensing technologies and experimental platforms need to be used in tandem. These technologies and platforms are discussed in this review, with a special focus on passive delivery systems in size range from a few 100 µm to a few mm. Challenges associated with characterizing these systems and evaluating their effect on oral drug delivery in the preclinical phase are also discussed.
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Sistemas de Liberación de Medicamentos , Desarrollo de Medicamentos/métodos , Tecnología Farmacéutica/métodos , Administración Oral , Animales , Disponibilidad Biológica , Preparaciones de Acción Retardada , Portadores de Fármacos/química , Liberación de Fármacos , Humanos , Tamaño de la Partícula , Preparaciones Farmacéuticas/administración & dosificación , Preparaciones Farmacéuticas/químicaRESUMEN
Bacterial biofilm-related infections are difficult to eradicate and require repeated treatments with high doses of antibiotics. Thus, there is an urgent need for new treatment strategies that minimize the use of antibiotics while enhancing biofilm eradication. Functionalized reservoir-based microdevices, such as, microcontainers (MCs), offer, high drug loading capacity, mucus embedment, and tuneable drug release. Here, MCs are loaded with the antibiotic ciprofloxacin (CIP), and sealed with a lid consisting of chitosan (CHI) and a mucolytic agent, N-acetylcysteine (NAC). It is found that CHI and NAC work synergistically, showing improved mucoadhesive and mucolytic properties. To better mimic the in vivo habitat of Pseudomonas aeruginosa (P. aeruginosa), the biofilm is grown in a mucin-containing medium on a newly developed centrifugal microfluidic system. The CHI/NAC coated MCs improve eradication of biofilm (88.22 ± 2.89%) compared to CHI-coated MCs (72.68 ± 3.73%) or bolus injection (39.86 ± 13.28%). The findings suggest that MCs are significantly more efficient than a bolus treatment. Furthermore, CHI/NAC functionalized MCs kill most of the biomass already after 5 h (80.75 ± 3.50%), mainly due to a fast drug release. This is the first time that CHI/NAC has been combined as a coating to explore mucolytic properties on bacterial biofilms.
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Antibacterianos , Mucinas , Antibacterianos/farmacología , Antibacterianos/uso terapéutico , Biopelículas , Pruebas de Sensibilidad Microbiana , Pseudomonas aeruginosaRESUMEN
Microscale devices are promising tools to overcome specific challenges within oral drug delivery. Despite the availability of advanced high-quality imaging techniques, visualization and tracking of microscale devices in the gastrointestinal (GI) tract is still a challenge. This work explores the possibilities of applying planar X-ray imaging and computed tomography (CT) scanning for visualization and tracking of microscale devices in the GI tract of rats. Microcontainers (MCs) are an example of microscale devices that have shown great potential as an oral drug delivery system. Barium sulfate (BaSO4) loaded into the cavity of the MCs increases their overall X-ray contrast, which allows them to be easily tracked. The BaSO4-loaded MCs are quantitatively tracked throughout the entire GI tract of rats by planar X-ray imaging and visualized in 3D by CT scanning. The majority of the BaSO4-loaded MCs are observed to retain in the stomach for 0.5-2 h, enter the cecum after 3-4 h, and leave the cecum and colon 8-10 h post-administration. The imaging approaches can be adopted and used with other types of microscale devices when investigating GI behavior in, for example, preclinical trials and potential clinical studies.
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Preparaciones Farmacéuticas , Tomografía Computarizada por Rayos X , Administración Oral , Animales , Sistemas de Liberación de Medicamentos , Tracto Gastrointestinal/diagnóstico por imagen , Ratas , Rayos XRESUMEN
For a better understanding of the effect of drugs and their interaction with cells and tissues, there is a need for in vitro and ex vivo model systems which enables studying these events. There are several in vitro methods available to evaluate the antioxidant activity; however, these methods do not factor in the complex in vivo physiology. Here we present an intestinal tissue modified oxygen electrode, used for the detection of the antioxidant effect of orally administered drugs in the presence of H2O2. Antioxidants are essential in the defense against oxidative stress, more specifically against reactive oxygen species such as H2O2. Due to the presence of native catalase in the intestine, with the tissue-based biosensor we were able to detect H2O2 in the range between 50 and 500 µM. The reproducibility of the sensor based on the calculated relative standard deviations was 15 ± 6%. We found that the O2 production by catalase from H2O2 was reduced in the presence of a well-known antioxidant, quinol. This indirectly detected antioxidant activity was also observed in the case of orally administered drugs with a reported anti-inflammatory effect such as mesalazine and paracetamol, while no antioxidant activity was recorded with aspirin and metformin.
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Antioxidantes/farmacología , Técnicas Biosensibles/métodos , Intestinos/efectos de los fármacos , Administración Oral , Animales , Catalasa/metabolismo , Peróxido de Hidrógeno/metabolismo , Ratones , Estrés Oxidativo/efectos de los fármacos , Oxígeno/metabolismoRESUMEN
Pathogenic bacterial biofilms can be life-threatening, greatly decrease patient's quality of life, and are a substantial burden on the healthcare system. Current methods for evaluation of antibacterial treatments in clinics and in vitro systems used in drug development and screening either do not facilitate biofilm formation or are cumbersome to operate, need large reagent volumes, and are costly, limiting their usability. To address these issues, this work presents the development of a robust in vitro cell culture platform compatible with confocal microscopy. The platform shaped as a compact disc facilitates long-term bacterial culture without external pumps and tubing and can be operated for several days without additional liquid handling. As an example, Pseudomonas aeruginosa biofilm is grown from single cells, and it is shown that (1) the platform delivers reproducible and reliable results; (2) growth is dependent on flow rate and growth medium composition; and (3) efficacy of antibiotic treatment depends on the formed biofilm. This platform enables biofilm growth, quantification, and treatment as in a conventional flow setup while decreasing the application barrier of lab-on-chip systems. It provides an easy-to-use, affordable option for end users working with cell culturing in relation to, e.g., diagnostics and drug screening.
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Antibacterianos/farmacología , Técnicas de Cultivo Celular por Lotes/métodos , Biopelículas/efectos de los fármacos , Dispositivos Laboratorio en un Chip , Microscopía Confocal/métodos , Pseudomonas aeruginosa , Técnicas de Cultivo Celular por Lotes/instrumentación , Biopelículas/crecimiento & desarrollo , Biomasa , Pseudomonas aeruginosa/fisiologíaRESUMEN
With the growing popularity and application of microfabricated devices in oral drug delivery (ODD), masking technologies for drug loading and surface modification become highly relevant. Considering the speed of design and fabrication processes and the necessity for continuous alterations of e.g. the shape and sizes of the devices during the optimization process, there is a need for adaptable, precise and low-cost masking techniques. Here, a novel method is presented for masking ODD microdevices, namely microcontainers, using the physical characteristics of polydimethylsiloxane (PDMS). When compared to a rigid microfabricated shadow mask, used for filling drugs in microcontainers, the PDMS masking technique allows more facile and precise loading of higher quantities of an active compound, without the need of alignment. The method provides flexibility and is adjustable to devices fabricated from different materials with various geometries, topologies and dimensions. This user-friendly flexible masking method overcomes the limitations of other masking techniques and is certainly not limited to ODD and is recommended for a wide range of microdevices.
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Dimetilpolisiloxanos/química , Sistemas de Liberación de Medicamentos/instrumentación , Dispositivos Laboratorio en un Chip , Fenómenos Mecánicos , Administración Oral , Diseño de EquipoRESUMEN
Evaluation and understanding the effect of drug delivery in in vitro systems is fundamental in drug discovery. We present an assay based on real-time electrical impedance spectroscopy (EIS) measurements that can be used to follow the internalisation and cytotoxic effect of a matrix metalloproteinase (MMP)-sensitive liposome formulation loaded with oxaliplatin (OxPt) on colorectal cancer cells. The EIS response identified two different cellular processes: (i) a negative peak in the cell index (CI) within the first 5 h, due to onset of liposome endocytosis, followed by (ii) a subsequent CI increase, due to the reattachment of cells until the onset of cytotoxicity with a decrease in CI. Free OxPt or OxPt-loaded Stealth liposomes did not show this two-stage EIS response; the latter can be due to the fact that Stealth cannot be cleaved by MMPs and thus is not taken up by the cells. Real-time bright-field imaging supported the EIS data, showing variations in cell adherence and cell morphology after exposure to the different liposome formulations. A drastic decrease in cell coverage as well as rounding up of cells during the first 5 h of exposure to OxPt-loaded (MMP)-sensitive liposome formulation is reflected by the first negative EIS response, which indicates the onset of liposome endocytosis. Graphical abstract.
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Antineoplásicos/administración & dosificación , Endocitosis , Liposomas , Oxaliplatino/administración & dosificación , Antineoplásicos/farmacología , Línea Celular Tumoral , Neoplasias Colorrectales/tratamiento farmacológico , Espectroscopía Dieléctrica , Humanos , Oxaliplatino/farmacologíaRESUMEN
An increased interest in colonic drug delivery has led to a higher focus on the design of delivery devices targeting this part of the gastrointestinal tract. Microcontainers have previously facilitated an increase in oral bioavailability of drugs. The surface texture and shape of microcontainers have proven to influence the mucoadhesion ex vivo. In the present work, these findings were further investigated using an in situ closed-loop perfusion technique in the rat colon, which allowed for simultaneous evaluation of mucoadhesion of the microcontainers as well as drug absorption. Cylindrical, triangular and cubic microcontainers, with the same exterior surface area, were evaluated based on in vitro release, in situ mucoadhesion and in situ absorption of amoxicillin. Additionally, the mucoadhesion of empty cylindrical microcontainers with and without pillars on the top surface was investigated. From the microscopy analysis of the colon sections after the in situ study, it was evident that a significantly higher percentage of cubic microcontainers than cylindrical microcontainers adhered to the intestinal mucus. Furthermore, the absorption rate constants and blood samples indicated that amoxicillin in cubic microcontainers was absorbed more readily than when cylindrical or triangular microcontainers were dosed. This could be due to a higher degree of mucoadhesion for these particular microcontainers.
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Reusability of sensors is relevant when aiming to decrease variation between measurements, as well as cost and time of analysis. We present an electrochemically assisted surface-enhanced Raman spectroscopy (SERS) platform with the capability to reverse the analyte-surface interaction, without damaging the SERS substrate, allowing for efficient sensor reuse. The platform was used in combination with a sample pretreatment step, when detecting melamine from milk. We found that the electrochemically enhanced analyte-surface interaction results in significant improvement in detection sensitivity, with detection limits (0.01 ppm in PBS and 0.3 ppm in milk) below the maximum allowed levels in food samples. The reversibility of interaction enabled continuous measurement in aqueous solution and a complete quantitative assay on a single SERS substrate.
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Leche/química , Triazinas/análisis , Animales , Bovinos , Técnicas Electroquímicas , Espectrometría RamanRESUMEN
Considering the widespread contaminations of food products with mycotoxins, it is important to develop, robust, time- and cost-effective detection methods. We developed and optimized an immunoassay using a continuous flow system for the detection of zearalenone (ZEN). The assay was performed in a flow mode using an automated sequential injection system. Time for an assay cycle was 18 Min. Under optimal conditions, the limit for quantification for ZEN was 0.40 µg L-1 with a linear dependency between concentration and signal amplitude between 0.10 and 10 µg L-1 . The assay proved to be robust and reliable with 13% relative standard deviation between measurements. By dissociating the antigen-antibody complex using a regeneration solution, we showed 50 times reusability of the immobilized antibodies without affecting the antigen-binding properties.