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The demand for continuous monitoring of biochemical markers for diagnostic purposes is increasing as it overcomes the limitations of traditional intermittent measurements. This study introduces a method for long-term, continuous plasmonic biosensing of oligonucleotides with high temporal resolution. Our method is based on a regeneration-based reversibility approach that ensures rapid reversibility in less than 1 minute, allowing the sensor to fully reset after each measurement. We investigated label-free and AuNP enhancements for different dynamic ranges and sensitivities, achieving a limit of detection down to pM levels. We developed a regeneration-based reversibility approach for continuous biosensing, optimizing buffer conditions using the Taguchi method to achieve rapid, consistent reversibility, ensuring reliable performance for long-term monitoring. We detected oligonucleotides in buffered and complex solutions, including undiluted and unfiltered human serum, for up to 100 sampling cycles in a day. Moreover, we showed the long-term stability of the sensor for monitoring capabilities in buffered solutions and human serum, with minimal signal value drift and excellent sensor reversibility for up to 9 days. Our method opens the door to new prospects in continuous biosensing by providing insights beyond intermittent measurements for numerous analytical and diagnostic applications.
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Organoid tumor models have emerged as a powerful tool in the fields of biology and medicine as such 3D structures grown from tumor cells recapitulate better tumor characteristics, making these tumoroids unique for personalized cancer research. Assessment of their functional behavior, particularly protein secretion, is of significant importance to provide comprehensive insights. Here, a label-free spectroscopic imaging platform is presented with advanced integrated optofluidic nanoplasmonic biosensor that enables real-time secretion analysis from single tumoroids. A novel two-layer microwell design isolates tumoroids, preventing signal interference, and the microarray configuration allows concurrent analysis of multiple tumoroids. The dual imaging capability combining time-lapse plasmonic spectroscopy and bright-field microscopy facilitates simultaneous observation of secretion dynamics, motility, and morphology. The integrated biosensor is demonstrated with colorectal tumoroids derived from both cell lines and patient samples to investigate their vascular endothelial growth factor A (VEGF-A) secretion, growth, and movement under various conditions, including normoxia, hypoxia, and drug treatment. This platform, by offering a label-free approach with nanophotonics to monitor tumoroids, can pave the way for new applications in fundamental biological studies, drug screening, and the development of therapies.
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Fator A de Crescimento do Endotélio Vascular , Humanos , Linhagem Celular Tumoral , Fator A de Crescimento do Endotélio Vascular/metabolismo , Fator A de Crescimento do Endotélio Vascular/genética , Técnicas Biossensoriais/métodos , Organoides/metabolismo , Neoplasias Colorretais/metabolismoRESUMO
Methods for the analysis of cell secretions at the single-cell level only provide semiquantitative endpoint readouts. Here we describe a microwell array for the real-time spatiotemporal monitoring of extracellular secretions from hundreds of single cells in parallel. The microwell array incorporates a gold substrate with arrays of nanometric holes functionalized with receptors for a specific analyte, and is illuminated with light spectrally overlapping with the device's spectrum of extraordinary optical transmission. Spectral shifts in surface plasmon resonance resulting from analyte-receptor bindings around a secreting cell are recorded by a camera as variations in the intensity of the transmitted light while machine-learning-assisted cell tracking eliminates the influence of cell movements. We used the microwell array to characterize the antibody-secretion profiles of hybridoma cells and of a rare subset of antibody-secreting cells sorted from human donor peripheral blood mononuclear cells. High-throughput measurements of spatiotemporal secretory profiles at the single-cell level will aid the study of the physiological mechanisms governing protein secretion.
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Leucócitos Mononucleares , Humanos , HibridomasRESUMO
Proteins secreted by cells play significant roles in mediating many physiological, developmental, and pathological processes due to their functions in intra/intercellular communication and signaling. Conventional end-point methods are insufficient for understanding the temporal response in cell secretion process, which is often highly dynamic. Furthermore, cellular heterogeneity makes it essential to analyze secretory proteins from single cells. To uncover individual cellular activities and the underlying kinetics, new technologies are needed for real-time analysis of the secretomes of many cells at single-cell resolution. This study reports a high-throughput biosensing microarray platform, which is capable of label-free and real-time secretome monitoring from a large number of living single cells using a biochip integrating ultrasensitive nanoplasmonic substrate and microwell compartments having volumes of â¼0.4 nL. Precise synchronization of image acquisition and microscope stage movement of the developed optical platform enables spectroscopic analysis with high temporal and spectral resolution. In addition, our system allows simultaneous optical imaging of cells to track morphology changes for a comprehensive understanding of cellular behavior. We demonstrated the platform performance by detecting interleukin-2 secretion from hundreds of single lymphoma cells in real-time over many hours. Significantly, the analysis of the secretion kinetics allows us to study cellular response to the stimulations in a statistical way. The new platform is a promising tool for the characterization of single-cell functionalities given its versatility, throughput and label-free configuration.
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Técnicas Biossensoriais , Técnicas Biossensoriais/métodos , Análise em MicrossériesRESUMO
Artificial cavitation as a prerequisite of sonoporation, plays an important role on the ultrasound (US) assisted drug delivery systems. In this study, we have proposed a new method of microbubble (MB) generation by local electrolysis of the medium. An integrated interdigital array of three-electrode system was designed and patterned on a nickel-coated quartz substrate and then, a short DC electrical pulse was applied that consequently resulted in distributed generation of microbubbles at the periphery of the electrodes. Growth of the carbon nanotube (CNT) nanostructures on the surface of the electrodes approximately increased the number of generated microbubbles up to 7-fold and decreased their average size from â¼20 µm for bare to â¼7 µm for CNT electrodes. After optimizing the three-electrode system, biocompatibility assays of the CNT electrodes stimulated by DC electrical micropulses were conducted. Finally, the effect of the proposed method on the sonoporation efficiency and drug uptake of breast cells were assessed using cell cycle and Annexin V/PI flow cytometry analysis. These results show the potential of electrochemical generation of MBs by CNT electrodes as an easy, available and promising technique for artificial cavitation and ultrasound assisted drug delivery.
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Antineoplásicos/química , Antineoplásicos/farmacologia , Nanotubos de Carbono/química , Neoplasias/tratamento farmacológico , Permeabilidade/efeitos dos fármacos , Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Sistemas de Liberação de Medicamentos/métodos , Eletrodos , Humanos , MicrobolhasRESUMO
It is increasingly being accepted that cells' physiological functions are substantially dependent on the mechanical characteristics of their surrounding tissue. This is mainly due to the key role of biomechanical forces on cells and their nucleus' shapes, which have the capacity to regulate chromatin conformation and thus gene regulations. Therefore, it is reasonable to postulate that altering the biomechanical properties of tissue may have the capacity to change cell functions. Here, the role of cell stretching (as a model of biomechanical variations) is probed in cell migration and invasion capacity using human normal and cancerous breast cells. By several analyses (i.e., scratch assay, invasion to endothelial barrier, real-time RNA sequencing, confocal imaging, patch clamp, etc.), it is revealed that the cell-stretching process could increase the migration and invasion capabilities of normal and cancerous cells, respectively. More specifically, it is found that poststretched breast cancer cells are found in low grades of invasion; they substantially upregulate the expression of manganese-dependent superoxide dismutase (MnSOD) through activation of H-Ras proteins, which subsequently induce aerobic glycolysis followed by an overproduction of matrix metalloproteinases (MMP)-reinforced filopodias. Presence of such invadopodias facilitates targeting of the endothelial layer, and increased invasive behaviors in breast cells are observed.
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Neoplasias da Mama/metabolismo , Regulação Neoplásica da Expressão Gênica , Glicólise , Proteínas de Neoplasias/biossíntese , Transdução de Sinais , Estresse Mecânico , Neoplasias da Mama/patologia , Feminino , Células Endoteliais da Veia Umbilical Humana , Humanos , Células MCF-7RESUMO
Detecting the micrometastasis is a major challenge in patients' survival. The small volume of the biopsied tissue results in limited number of histopathological samples and might reduce the rate of accurate diagnosis even by molecular technologies. We introduce a microelectronic biochip (named Metas-Chip) to detect the micrometastasis in unprocessed liquid or solid samples. It works based on the tendency of malignant cells to track single human umbilical vein endothelial cell (HUVEC)-sensing traps. Such cells detach themselves from the biopsied sample and invade the sensing traps by inducing membrane retraction and blebbing, which result in sharp changes in electrical response of the sensing elements. Metas-Chip identified the metastasis in more than 70 breast cancer patients, in less than 5 h. Moreover it detected the metastasis in lymph nodes of nine patients whom were missed by conventional pathological procedure. Multilevel IHC and real-time polymerase chain reaction (RT-PCR) tests confirmed the diagnosis.