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Through digital imaging, microscopy has evolved from primarily being a means for visual observation of life at the micro- and nano-scale, to a quantitative tool with ever-increasing resolution and throughput. Artificial intelligence, deep neural networks, and machine learning are all niche terms describing computational methods that have gained a pivotal role in microscopy-based research over the past decade. This Roadmap is written collectively by prominent researchers and encompasses selected aspects of how machine learning is applied to microscopy image data, with the aim of gaining scientific knowledge by improved image quality, automated detection, segmentation, classification and tracking of objects, and efficient merging of information from multiple imaging modalities. We aim to give the reader an overview of the key developments and an understanding of possibilities and limitations of machine learning for microscopy. It will be of interest to a wide cross-disciplinary audience in the physical sciences and life sciences.
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It is difficult to model in vitro the intestine when seeking to include crosstalk with the gut microbiota, immune and neuroendocrine systems. Here we present a roadmap of the current models to facilitate the choice in preclinical and translational research with a focus on gut-on-chip. These micro physiological systems (MPS) are microfluidic devices that recapitulate in vitro the physiology of the intestine. We reviewed the gut-on-chips that had been developed in academia and industries as single chip and that have three main purpose: replicate the intestinal physiology, the intestinal pathological features, and for pharmacological tests.
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The control of the morphology, as well as the physical and chemical properties, of nanopores is a key issue for many applications. Reducing pore size is important in nanopore-based sensing applications as it helps to increase sensitivity. Changes of other physical properties such as surface net charge can also modify transport selectivity of the pores. We have studied how polyelectrolyte layer-by-layer (LBL) surface modification can be used to change the characteristics of nanoporous membranes. Studies were performed with a custom made three-dimensional multilayer microfluidic device able to fit membrane samples. The device allowed us to efficiently control LBL film deposition over blank low-cost commercially available polycarbonate track-etched (PCTE) membranes. We have demonstrated pore diameter reduction and deposition of the layers inside the pores through confocal and SEM images. Posterior impedance measurement studies served to evaluate experimentally the effect of the LBL deposition on the net inner nanopore surface charge and diameter. Measurements using direct current (DC) and alternative current (AC) voltages have demonstrated contrasted behaviors depending on the number and parity of deposited opposite charge layers. PCTE membranes are originally negatively charged and results evidenced higher impedance increases for paired charge LBL depositions. Impedance decreased when an unpaired positive layer was added. These results showed a different influence on the overall ion motility due to the effect of different surface charges. Results have been fit into a model that suggested a strong dependence of nanopores' impedance module to surface charge on conductive buffers, such as Phosphate Buffer Saline (PBS), even on relatively large nanopores. In AC significant differences between paired and unpaired charged LBL depositions tended to disappear as the total number of layers increased.
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Ultrasmooth gold/silver/gold trilayer nanostructured plasmonic sensors were obtained using commercial Blu-ray optical discs as nanoslits-based flexible polymer substrates. A thin gold film was used as an adhesion and nucleation layer to improve the chemical stability and reduce the surface roughness of the overlying silver film, without increasing ohmic plasmon losses. The structures were physically and optically characterized and compared with nanostructures of single gold layer. Ultrasmooth and chemically stable trilayer nanostructures with a surface roughness <0.5 nm were obtained following a simple and reproducible fabrication process. They showed a figure of merit (FOM) value up to 69.2 RIU-1 which is significantly higher (more than 95%) than the gold monolayer counterpart. Their potential for biosensing was demonstrated by employing the trilayer sensor for the direct and refractometric (label-free) detection of C-reactive protein (CRP) biomarker in undiluted urine achieving a Limit of Detection (LOD) in the pM order.
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Ouro/química , Nanoestruturas/química , Cimento de Policarboxilato/química , Prata/química , Ressonância de Plasmônio de Superfície/métodos , Proteína C-Reativa/urina , Humanos , Limite de Detecção , Fenômenos ÓpticosRESUMO
Nanostructure-based plasmonic biosensors have quickly positioned themselves as interesting candidates for the design of portable optical biosensor platforms considering the potential benefits they can offer in integration, miniaturization, multiplexing, and real-time label-free detection. We have developed a simple integrated nanoplasmonic sensor taking advantage of the periodic nanostructured array of commercial Blu-ray discs. Sensors with two gold film thicknesses (50 and 100nm) were fabricated and optically characterized by varying the oblique-angle of the incident light in optical reflectance measurements. Contrary to the use normal light incidence previously reported with other optical discs, we observed an enhancement in sensitivity and a narrowing of the resonant linewidths as the light incidence angle was increased, which could be related to the generation of Fano resonant modes. The new sensors achieve a figure of merit (FOM) up to 35 RIU-1 and a competitive bulk limit of detection (LOD) of 6.3×10-6 RIU. These values significantly improve previously reported results obtained with normal light incidence reflectance measurements using similar structures. The sensor has been combined with versatile, simple, ease to-fabricate microfluidics. The integrated chip is only 1cm2 (including a PDMS flow cell with a 50µm height microfluidic channel fabricated with double-sided adhesive tape) and all the optical components are mounted on a 10cm×10cm portable prototype, illustrating its facile miniaturization, integration and potential portability. Finally, to assess the label-free biosensing capability of the new sensor, we have evaluated the presence of specific antibodies against the GTF2b protein, a tumor-associate antigen (TAA) related to colorectal cancer. We have achieved a LOD in the pM order and have assessed the feasibility of directly measuring biological samples such as human serum.
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Ouro/química , Técnicas Analíticas Microfluídicas/instrumentação , Nanoestruturas/química , Ressonância de Plasmônio de Superfície/instrumentação , Anticorpos/análise , Anticorpos/sangue , Neoplasias Colorretais/sangue , Neoplasias Colorretais/diagnóstico , Desenho de Equipamento , Humanos , Proteínas Imobilizadas/química , Limite de Detecção , Fator de Transcrição TFIIB/químicaRESUMO
Bacteria concentration and detection is time-consuming in regular microbiology procedures aimed to facilitate the detection and analysis of these cells at very low concentrations. Traditional methods are effective but often require several days to complete. This scenario results in low bioanalytical and diagnostic methodologies with associated increased costs and complexity. In recent years, the exploitation of the intrinsic electrical properties of cells has emerged as an appealing alternative approach for concentrating and detecting bacteria. The combination of dielectrophoresis (DEP) and impedance analysis (IA) in microfluidic on-chip platforms could be key to develop rapid, accurate, portable, simple-to-use and cost-effective microfluidic devices with a promising impact in medicine, public health, agricultural, food control and environmental areas. The present document reviews recent DEP and IA combined approaches and the latest relevant improvements focusing on bacteria concentration and detection, including selectivity, sensitivity, detection time, and conductivity variation enhancements. Furthermore, this review analyses future trends and challenges which need to be addressed in order to successfully commercialize these platforms resulting in an adequate social return of public-funded investments.
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Bactérias/metabolismo , Impedância Elétrica , Dispositivos Lab-On-A-Chip , Técnicas Analíticas Microfluídicas/instrumentação , Soluções Tampão , Transferência de TecnologiaRESUMO
The present paper reports a bacteria autonomous controlled concentrator prototype with a user-friendly interface for bench-top applications. It is based on a microfluidic lab-on-a-chip and its associated custom instrumentation, which consists of a dielectrophoretic actuator, to preconcentrate the sample, and an impedance analyzer, to measure concentrated bacteria levels. The system is composed of a single microfluidic chamber with interdigitated electrodes and an instrumentation with custom electronics. The prototype is supported by a real-time platform connected to a remote computer, which automatically controls the system and displays impedance data used to monitor the status of bacteria accumulation on-chip. The system automates the whole concentrating operation. Performance has been studied for controlled volumes of Escherichia coli samples injected into the microfluidic chip at constant flow rate of 10 µL/min. A media conductivity correcting protocol has been developed, as the preliminary results showed distortion of the impedance analyzer measurement produced by bacterial media conductivity variations through time. With the correcting protocol, the measured impedance values were related to the quantity of bacteria concentrated with a correlation of 0.988 and a coefficient of variation of 3.1%. Feasibility of E. coli on-chip automated concentration, using the miniaturized system, has been demonstrated. Furthermore, the impedance monitoring protocol had been adjusted and optimized, to handle changes in the electrical properties of the bacteria media over time.
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Eletroforese/instrumentação , Eletroforese/métodos , Escherichia coli/isolamento & purificação , Técnicas Analíticas Microfluídicas/instrumentação , Impedância Elétrica , Eletrodos , Desenho de Equipamento , Escherichia coli/fisiologia , Estudos de ViabilidadeRESUMO
Dielectrophoretic (DEP) manipulation of cells present in real samples is challenging. We show in this work that an interdigitated DEP chip can be used to trap and wash a population of the food-spoiling yeast Zygosaccharomyces rouxii that contaminates a sample of apple juice. By previously calibrating the chip, the yeast population loaded is efficiently trapped, washed, and recovered in a small-volume fraction that, in turn, can be used for efficient PCR detection of this yeast. DEP washing of yeast cells gets rid of PCR inhibitors present in apple juice and facilitates PCR analysis. This and previous works on the use of DEP chips to improve PCR analysis show that a potential use of DEP is to be used as a treatment of real samples prior to PCR.
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Bebidas/microbiologia , Eletroforese/instrumentação , Malus , Reação em Cadeia da Polimerase/instrumentação , Zygosaccharomyces/isolamento & purificação , Eletroforese/métodos , Reação em Cadeia da Polimerase/métodos , Zygosaccharomyces/químicaRESUMO
We describe a novel continuous-flow cell concentrator microdevice based on dielectrophoresis, and its associated custom-made control unit. The performances of a classical interdigitated metal electrode-based dielectrophoresis microfluidic device and this enhanced version, that includes insulator-based pole structures, were compared using the same setup. Escherichia coli samples were concentrated at several continuous flows and the device's trapping efficiencies were evaluated by exhaustive cell counts. Our results show that pole structures enhance the retention up to 12.6%, obtaining significant differences for flow rates up to 20 µL/min, when compared to an equivalent classical interdigitated electrodes setup. In addition, we performed a subsequent proteomic analysis to evaluate the viability of the biological samples after the long exposure to the actuating electrical field. No Escherichia coli protein alteration in any of the two systems was observed.
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Eletroforese/instrumentação , Técnicas Analíticas Microfluídicas/instrumentação , Desenho de Equipamento , Escherichia coli/isolamento & purificação , Proteômica/instrumentaçãoRESUMO
Electrokinetic techniques are contact-free methods currently used in many applications, where precise handling of biological entities, such as cells, bacteria or nucleic acids, is needed. These techniques are based on the effect of electric fields on molecules suspended in a fluid, and the corresponding induced motion, which can be tuned according to some known physical laws and observed behaviours. Increasing interest on the application of such strategies in order to improve the detection of DNA strands has appeared during the recent decades. Classical electrode-based DNA electrochemical biosensors with combined electrokinetic techniques present the advantage of being able to improve the working electrode's bioactive part during their fabrication and also the hybridization yield during the sensor detection phase. This can be achieved by selectively manipulating, driving and directing the molecules towards the electrodes increasing the speed and yield of the floating DNA strands attached to them. On the other hand, this technique can be also used in order to make biosensors reusable, or reconfigurable, by simply inverting its working principle and pulling DNA strands away from the electrodes. Finally, the combination of these techniques with nanostructures, such as nanopores or nanochannels, has recently boosted the appearance of new types of electrochemical sensors that exploit the time-varying position of DNA strands in order to continuously scan these molecules and to detect their properties. This review gives an insight into the main forces involved in DNA electrokinetics and discusses the state of the art and uses of these techniques in recent years.
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Técnicas Biossensoriais/métodos , DNA/análise , Técnicas Eletroquímicas/métodos , Dispositivos Lab-On-A-Chip , Algoritmos , DNA/química , DNA/isolamento & purificação , Nanoporos , ViscosidadeRESUMO
The objective of the present work was to detect and analyze wheezes by means of a highly sensitive time-frequency algorithm. Automatic measurements were compared with clinical auscultation for forced exhalation segments from 1.2 to 0 liters/second (l/s). Sensitivities between 100% and 71%, as a function of flow level related to wheezing segments detection, were achieved. Time-frequency wheeze parameters were measured for the flow range from 1.2 to 0.2 l/s. Wheezes were detected in both analyzed groups; asthmatics (N = 16) and control subjects (N = 15). Significant differences between groups were found for the mean number of wheezes detected at basal condition (p = 0.0003). Frequency parameter differences were also significant (0.0112 < p < 0.0307). All these parameters were also studied after applying a bronchodilator drug (Terbutaline). Significant differences between patient groups were found when studying the changes in the number of wheezes for each patient (p = 0.0195). Finally, limited bandwidth parameters, which measure the bronchodilator response, were also studied.