RESUMO
The ability to deploy decentralized laboratories with autonomous and reliable disease diagnosis holds the potential to deliver accessible healthcare services for public safety. While microfluidic technologies provide precise manipulation of small fluid volumes with improved assay performance, their limited automation and versatility confine them to laboratories. Herein, we report the utility of multicolor assay-on-a-chip processed by robotic operation (MACpro), to address this unmet need. The MACpro platform comprises a robot-microfluidic interface and an eye-in-hand module that provides flexible yet stable actions to execute tasks in a programmable manner, such as the precise manipulation of the microfluidic chip along with different paths. Notably, MACpro shows improved detection performance by integrating the microbead-based antibody immobilization with enhanced target recognition and multicolor sensing via Cu2+-catalyzed plasmonic etching of gold nanorods for rapid and sensitive analyte quantification. Using interferon-gamma as an example, we demonstrate that MACpro completes a sample-to-answer immunoassay within 30 min and achieves a 10-fold broader dynamic range and a 10-fold lower detection limit compared to standard enzyme-linked immunosorbent assays (0.66 vs 5.2 pg/mL). MACpro extends the applications beyond traditional laboratories and presents an automated solution to expand diagnostic capacity in diverse settings.
Assuntos
Dispositivos Lab-On-A-Chip , Robótica , Humanos , Imunoensaio/métodos , Interferon gama/análise , Técnicas Analíticas Microfluídicas/instrumentação , Ouro/químicaRESUMO
Protein networks can be assembled in vitro for basic biochemistry research, drug screening, and the creation of artificial cells. Two standard methodologies are used: manual pipetting and pipetting robots. Manual pipetting has limited throughput in the number of input reagents and the combination of reagents in a single sample. While pipetting robots are evident in improving pipetting efficiency and saving hands-on time, their liquid handling volume usually ranges from a few to hundreds of microliters. Microfluidic methods have been developed to minimize the reagent consumption and speed up screening but are challenging in multifactorial protein studies due to their reliance on complex structures and labeling dyes. Here, we engineered a new impact-printing-based methodology to generate printed microdroplet arrays containing water-in-oil droplets. The printed droplet volume was linearly proportional (R2 = 0.9999) to the single droplet number, and each single droplet volume was around 59.2 nL (coefficient of variation = 93.8%). Our new methodology enables the study of protein networks in both membrane-unbound and -bound states, without and with anchor lipids DGS-NTA(Ni), respectively. The methodology is demonstrated using a subnetwork of mitogen-activated protein kinase (MAPK). It takes less than 10 min to prepare 100 different droplet-based reactions, using <1 µL reaction volume at each reaction site. We validate the kinase (ATPase) activity of MEK1 (R4F)* and ERK2 WT individually and together under different concentrations, without and with the selective membrane attachment. Our new methodology provides a reagent-saving, efficient, and flexible way for protein network research and related applications.
Assuntos
Técnicas Analíticas Microfluídicas , Microfluídica , Avaliação Pré-Clínica de Medicamentos , Técnicas Analíticas Microfluídicas/métodos , Microfluídica/métodos , Impressão Tridimensional , Água/químicaRESUMO
Enzyme-linked immunosorbent assays (ELISA), as one of the most used immunoassays, have been conducted ubiquitously in hospitals, research laboratories, etc. However, the conventional ELISA procedure is usually laborious, occupies bulky instruments, consumes lengthy operation time, and relies considerably on the skills of technicians, and such limitations call for innovations to develop a fully automated ELISA platform. In this paper, we have presented a system incorporating a robotic-microfluidic interface (RoMI) and a modular hybrid microfluidic chip that embeds a highly sensitive nanofibrous membrane, referred to as the Robotic ELISA, to achieve human-free sample-to-answer ELISA tests in a fully programmable and automated manner. It carries out multiple bioanalytical procedures to replace the manual steps involved in classic ELISA operations, including the pneumatically driven high-precision pipetting, efficient mixing and enrichment enabled by back-and-forth flows, washing, and integrated machine vision for colorimetric readout. The Robotic ELISA platform has achieved a low limit of detection of 0.1 ng/mL in the detection of a low sample volume (15 µL) of chloramphenicol within 20 min without human intervention, which is significantly faster than that of the conventional ELISA procedure. Benefiting from its modular design and automated operations, the Robotic ELISA platform has great potential to be deployed for a broad range of detections in various resource-limited settings or high-risk environments, where human involvement needs to be minimized while the testing timeliness, consistency, and sensitivity are all desired.
Assuntos
Técnicas Analíticas Microfluídicas , Procedimentos Cirúrgicos Robóticos , Colorimetria , Ensaio de Imunoadsorção Enzimática , Humanos , Imunoensaio , MicrofluídicaRESUMO
Multi-drug resistance (MDR) is a curious bottleneck in cancer research and chemotherapy, whereby some cells rapidly adapt to the tumor microenvironment via a myriad of heterogeneous metabolic activities. Despite being a major impediment to treatment, there is a silver lining: control over metabolic regulation could be an effective approach to overcome or correct resistance pathways. In this critical review, we comprehensively and carefully curated and analyzed large networks of previously identified proteins associated with metabolic adaptation in MDR. We employed data and text mining to study and categorize more than 600 studies in PubMed, with particular focus on AMPK, a central and fundamental modulator in the energy metabolism network that has been specifically implicated in cancer MDR pathways. We have identified one protein set of metabolic adaptations with 137 members closely related to cancer MDR processes, and a second protein set with 165 members derived from AMPK-based networks, with 28 proteins found at the intersection between the two sets. Furthermore, according to genomics analysis of the cancer genome atlas (TCGA) provisional data, the highest alteration frequency (80.0%) of the genes encoding the intersected proteins (28 proteins), ranked three cancer types with quite remarkable significance across 166 studies. The hierarchical relationships of the entire identified gene and protein networks indicate broad correlations in AMPK-mediated metabolic regulation pathways, which we use decipher and depict the metabolic roles of AMPK and demonstrate the potential of metabolic control for therapeutic intervention in MDR.
Assuntos
Proteínas Quinases Ativadas por AMP/metabolismo , Resistência a Múltiplos Medicamentos , Resistencia a Medicamentos Antineoplásicos , Metabolismo Energético , Neoplasias/metabolismo , Animais , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Biomarcadores Tumorais , Suscetibilidade a Doenças , Resistencia a Medicamentos Antineoplásicos/genética , Humanos , Neoplasias/tratamento farmacológico , Neoplasias/etiologia , Neoplasias/patologiaRESUMO
Assembly of recombinant multiprotein systems requires multiple culturing and purification steps that scale linearly with the number of constituent proteins. This problem is particularly pronounced in the preparation of the 34 proteins involved in transcription and translation systems, which are fundamental biochemistry tools for reconstitution of cellular pathways ex vivo. Here, we engineer synthetic microbial consortia consisting of between 15 and 34 Escherichia coli strains to assemble the 34 proteins in a single culturing, lysis, and purification procedure. The expression of these proteins is controlled by synthetic genetic modules to produce the proteins at the correct ratios. We show that the pure multiprotein system is functional and reproducible, and has low protein contaminants. We also demonstrate its application in the screening of synthetic promoters and protease inhibitors. Our work establishes a novel strategy for producing pure translation machinery, which may be extended to the production of other multiprotein systems.
Assuntos
Proteínas de Escherichia coli/genética , Escherichia coli/genética , Consórcios Microbianos/genética , Engenharia de Proteínas/métodos , Proteínas Recombinantes/biossíntese , Biologia Sintética/métodos , Biossíntese de Proteínas , Proteínas Recombinantes/genéticaRESUMO
Wearable biosensors as a user-friendly measurement platform have become a rapidly growing field of interests due to their possibility in integrating traditional medical diagnostics and healthcare management into miniature lab-on-body analytic devices. This paper demonstrates a flexible and skin-mounted band that combines superhydrophobic-superhydrophilic microarrays with nanodendritic colorimetric biosensors toward in situ sweat sampling and analysis. Particularly, on the superwettable bands, the superhydrophobic background could confine microdroplets into superhydrophilic microwells. On-body investigations further reveal that the secreted sweat is repelled by the superhydrophobic silica coating and precisely collected and sampled onto the superhydrophilic micropatterns with negligible lateral spreading, which provides an independent "vessel" toward cellphone-based sweat biodetection (pH, chloride, glucose and calcium). Such wearable, superwettable band-based biosensors with improved interface controllability could significantly enhance epidemical sweat sampling in well-defined sites, holding a great promise for facile and noninvasive biofluids analysis.
Assuntos
Técnicas Biossensoriais/métodos , Cálcio/análise , Cloretos/análise , Glucose/análise , Polietilenotereftalatos/química , Suor/química , Técnicas Biossensoriais/instrumentação , Telefone Celular , Colorimetria/instrumentação , Colorimetria/métodos , Humanos , Concentração de Íons de Hidrogênio , Interações Hidrofóbicas e Hidrofílicas , Análise em Microsséries/instrumentação , Análise em Microsséries/métodos , Nanoestruturas/química , Maleabilidade , Dióxido de Silício/química , MolhabilidadeRESUMO
The United States is currently experiencing an opioid crisis, with more than 47,000 deaths in 2017 due to opioid overdoses. Current approaches for opioid identification and quantification in body fluids include immunoassays and chromatographic methods (e.g., LC-MS, GC-MS), which require expensive instrumentation and extensive sample preparation. Our aim was to develop a portable point-of-care device that can be used for the instant detection of opioids in body fluids. Here, we reported the development of a morphine-sensitive fluorescence-based sensor chip to sensitively detect morphine in the blood using a homogeneous immunoassay without any washing steps. Morphine-sensitive illuminating peptides were identified using a high throughput one-bead one-compound (OBOC) combinatorial peptide library approach. The OBOC libraries contain a large number of random peptides with a molecular rotor dye, malachite green (MG), that are coupled to the amino group on the side chain of lysine at different positions of the peptides. The OBOC libraries were then screened for fluorescent activation under a confocal microscope, using an anti-morphine monoclonal antibody as the screening probe, in the presence and absence of free morphine. Using this novel three-step fluorescent screening assay, we were able to identify the peptide-beads that fluoresce in the presence of an anti-morphine antibody, but lost fluorescence when the free morphine was present. After the positive beads were decoded using automatic Edman microsequencing, the morphine-sensitive illuminating peptides were then synthesized in soluble form, functionalized with an azido group, and immobilized onto microfabricated PEG-array spots on a glass slide. The sensor chip was then evaluated for the detection of morphine in plasma. We demonstrated that this proof-of-concept platform can be used to develop fluorescence-based sensors against morphine. More importantly, this technology can also be applied to the discovery of other novel illuminating peptidic sensors for the detection of illicit drugs and cancer biomarkers in body fluids.
Assuntos
Analgésicos Opioides/análise , Analgésicos Opioides/sangue , Líquidos Corporais/química , Técnicas de Química Combinatória/métodos , Morfina/análise , Morfina/sangue , Peptídeos/química , Cromatografia Líquida , Ensaios de Triagem em Larga Escala , Humanos , Biblioteca de PeptídeosRESUMO
Bioinspired superwettable micropatterns that combine superhydrophobicity and superhydrophilicity have been proved to exhibit outstanding capacity in controlling and patterning microdroplets and possessed new functionalities and possibilities in emerging sensing applications. Here, we introduce a flexible tape-based superhydrophilic-superhydrophobic tape toward on-site heavy metals monitoring. On such a superwettable tape, capillarity-assisted superhydrophilic microwells allow directly anchoring indicators in fixed locations and sampling into a test zone via simple dip-pull from an origin specimen solution. In contrast, the superhydrophobic substrate could confine the microdroplets in the superhydrophilic microwells for reducing the amount of analytical solution. The tape-based microchip also displays excellent flexibility against stretching, bending, and torquing for expanding wearable and portable sensing devices. Qualitative and quantitative colorimetric assessments of multiplex heavy metal analyses (chromium, copper, and nickel) by the naked eye are also achieved. The superwettable tape-based platforms with a facile operation mode and accessible signal read-out represent unrevealed potential for on-site environmental monitoring.
Assuntos
Colorimetria/métodos , Metais Pesados/análise , Cromo/análise , Cobre/análise , Monitoramento Ambiental , Interações Hidrofóbicas e Hidrofílicas , Níquel/análise , Imagem com Lapso de TempoRESUMO
In this paper, we introduce a novel microfluidic combinatorial synthesis platform, referred to as Microfluidic Print-to-Synthesis (MPS), for custom high-throughput and automated synthesis of a large number of unique peptides in a microarray format. The MPS method utilizes standard Fmoc chemistry to link amino acids on a polyethylene glycol (PEG)-functionalized microdisc array. The resulting peptide microarrays permit rapid screening for interactions with molecular targets or live cells, with low nonspecific binding. Such combinatorial peptide microarrays can be reliably prepared at a spot size of 200 µm with 1 mm center-to-center distance, dimensions that require only minimal reagent consumption (less than 30 nL per spot per coupling reaction). The MPS platform has a scalable design for extended multiplexibility, allowing for 12 different building blocks and coupling reagents to be dispensed in one microfluidic cartridge in the current format, and could be further scaled up. As proof of concept for the MPS platform, we designed and constructed a focused tetrapeptide library featuring 2560 synthetic peptide sequences, capped at the N-terminus with 4-[( N'-2-methylphenyl)ureido]phenylacetic acid. We then used live human T lymphocyte Jurkat cells as a probe to screen the peptide microarrays for their interaction with α4ß1 integrin overexpressed and activated on these cells. Unlike the one-bead-one-compound approach that requires subsequent decoding of positive beads, each spot in the MPS array is spatially addressable. Therefore, this platform is an ideal tool for rapid optimization of lead compounds found in nature or discovered from diverse combinatorial libraries, using either biochemical or cell-based assays.
Assuntos
Técnicas de Química Combinatória , Técnicas Analíticas Microfluídicas , Peptídeos/análise , Impressão , Análise Serial de Proteínas , Humanos , Células Jurkat , Tamanho da Partícula , Biblioteca de PeptídeosRESUMO
Traditional high-throughput drug combination screening requires automatic pipetting of drugs into high-density microtiter plates. Here, a drug-on-pillar platform is proposed for efficient combination drug screening. Using the proposed approach, combination drug screening can be carried out in a plug-and-play manner, allowing for high-throughput screening of large permutations of drug combinations at various concentrations, such that drug dispensing and cell-based screening can be temporally separated and therefore can potentially be performed at distant laboratories. The dispensing is implemented using our recently developed microfluidic pneumatic printing platform, which features a low-cost disposable cartridge that minimizes cross contamination. Moreover, our previously developed drug nanoformulation method with amphiphilic telodendrimers has been utilized to maintain drug stability in a dry form, allowing for convenient drug storage, shipping, and subsequent rehydration. Combining the features described above, we have implemented a 1260-spot drug combination array to study the effect of paired drugs against MDA-MB-231 triple negative human breast cancer cells. This study supports the feasibility of the drug-on-pillar platform for combination drug screening and has provided valuable insight into drug combination efficacy against breast cancer.
Assuntos
Antineoplásicos/farmacologia , Doxorrubicina/farmacologia , Técnicas Analíticas Microfluídicas , Impressão Tridimensional , Neoplasias de Mama Triplo Negativas/tratamento farmacológico , Antineoplásicos/química , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Relação Dose-Resposta a Droga , Doxorrubicina/química , Combinação de Medicamentos , Avaliação Pré-Clínica de Medicamentos , Ensaios de Seleção de Medicamentos Antitumorais , Humanos , Relação Estrutura-Atividade , Neoplasias de Mama Triplo Negativas/patologia , Células Tumorais CultivadasRESUMO
When a constraint is removed, confluent cells migrate directionally into the available space. How the migration directionality and speed increase are initiated at the leading edge and propagate into neighboring cells are not well understood. Using a quantitative visualization technique-Particle Image Velocimetry (PIV)-we revealed that migration directionality and speed had strikingly different dynamics. Migration directionality increases as a wave propagating from the leading edge into the cell sheet, while the increase in cell migration speed is maintained only at the leading edge. The overall directionality steadily increases with time as cells migrate into the cell-free space, but migration speed remains largely the same. A particle-based compass (PBC) model suggests cellular interplay (which depends on cell-cell distance) and migration speed are sufficient to capture the dynamics of migration directionality revealed experimentally. Extracellular Ca2+ regulated both migration speed and directionality, but in a significantly different way, suggested by the correlation between directionality and speed only in some dynamic ranges. Our experimental and modeling results reveal distinct directionality and speed dynamics in collective migration, and these factors can be regulated by extracellular Ca2+ through cellular interplay. Quantitative visualization using PIV and our PBC model thus provide a powerful approach to dissect the mechanisms of collective cell migration.
Assuntos
Cálcio/metabolismo , Comunicação Celular , Movimento Celular , Epitélio Corneano/citologia , Materiais Biocompatíveis/química , Contagem de Células , Linhagem Celular , Dimetilpolisiloxanos/química , Epitélio Corneano/metabolismo , Humanos , Modelos Biológicos , CicatrizaçãoRESUMO
Since the 1960s, combination chemotherapy has been widely utilized as a standard method to treat cancer. However, because of the potentially enormous number of drug candidates and combinations, conventional identification methods of the effective drug combinations are usually associated with significantly high operational costs, low throughput screening, laborious and time-consuming procedures, and ethical concerns. In this paper, we present a low-cost, high-efficiency microfluidic print-to-screen (P2S) platform, which integrates combinatorial screening with biomolecular printing for high-throughput screening of anticancer drug combinations. This P2S platform provides several distinct advantages and features, including automatic combinatorial printing, high-throughput parallel drug screening, modular disposable cartridge, and biocompatibility, which can potentially speed up the entire discovery cycle of potent drug combinations. Microfluidic impact printing utilizing plug-and-play microfluidic cartridges is experimentally characterized with controllable droplet volume and accurate positioning. Furthermore, the combinatorial print-to-screen assay is demonstrated in a proof-of-concept biological experiment which can identify the positive hits among the entire drug combination library in a parallel and rapid manner. Overall, this microfluidic print-to-screen platform offers a simple, low-cost, high-efficiency solution for high-throughput large-scale combinatorial screening and can be applicable for various emerging applications in drug cocktail discovery.
Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/análise , Técnicas de Química Combinatória , Avaliação Pré-Clínica de Medicamentos , Ensaios de Triagem em Larga Escala , Técnicas Analíticas Microfluídicas , Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Sobrevivência Celular/efeitos dos fármacos , Ensaios de Triagem em Larga Escala/instrumentação , Humanos , Técnicas Analíticas Microfluídicas/instrumentação , Impressão/instrumentação , Relação Estrutura-Atividade , Células Tumorais CultivadasRESUMO
Mechanisms that guide directional migration of neuroblasts from the subventricular zone (SVZ) are not well understood. We report here that endogenous electric currents serve as a guidance cue for neuroblast migration. We identify the existence of naturally occurring electric currents (1.5±0.6 µA/cm(2), average field strength of â¼3 mV/mm) along the rostral migration path in adult mouse brain. Electric fields of similar strength direct migration of neuroblasts from the SVZ in culture and in brain slices. The purinergic receptor P2Y1 mediates this migration. The results indicate that naturally occurring electric currents serve as a new guidance mechanism for rostral neuronal migration.
Assuntos
Movimento Celular , Células-Tronco Neurais/fisiologia , Receptores Purinérgicos P2Y1/fisiologia , Animais , Ventrículos Cerebrais/citologia , Impedância Elétrica , Estimulação Elétrica , Eletrodos , Técnicas de Silenciamento de Genes , Técnicas In Vitro , Camundongos , Células-Tronco Neurais/efeitos dos fármacos , Bulbo Olfatório/citologia , Antagonistas do Receptor Purinérgico P2Y/farmacologia , RNA Interferente Pequeno/genética , Suramina/farmacologiaRESUMO
The role that posterior parietal (PPC) and motor cortices play in modulating neural responses in somatosensory areas 1 and 2 was examined with reversible deactivation by transient cooling. Multiunit recordings from neurons in areas 1 and 2 were collected from six anesthetized adult monkeys (Macaca mulatta) before, during, and after reversible deactivation of areas 5L or 7b or motor cortex (M1/PM), while select locations on the hand and forelimb were stimulated. Response changes were quantified as increases and decreases to stimulus-driven activity relative to baseline and analyzed during three recording epochs: during deactivation ("cool") and at two time points after deactivation ("rewarm 1," "rewarm 2"). Although the type of response change observed was variable, for neurons at the recording sites tested >90% exhibited a significant change in response during cooling of 7b while cooling area 5L or M1/PM produced a change in 75% and 64% of sites, respectively. These results suggest that regions in the PPC, and to a lesser extent motor cortex, shape the response characteristics of neurons in areas 1 and 2 and that this kind of feedback modulation is necessary for normal somatosensory processing. Furthermore, this modulation appears to happen on a minute-by-minute basis and may serve as the substrate for phenomena such as somatosensory attention.
Assuntos
Mãos/fisiologia , Neurônios/fisiologia , Lobo Parietal/fisiologia , Percepção do Tato/fisiologia , Potenciais de Ação , Animais , Temperatura Baixa , Feminino , Macaca mulatta , Masculino , Microeletrodos , Atividade Motora/fisiologia , Córtex Motor/fisiologia , Estimulação Física , Córtex Somatossensorial/fisiologiaRESUMO
Somatosensory processing in the anesthetized macaque monkey was examined by reversibly deactivating posterior parietal areas 5L and 7b and motor/premotor cortex (M1/PM) with microfluidic thermal regulators developed by our laboratories. We examined changes in receptive field size and configuration for neurons in areas 1 and 2 that occurred during and after cooling deactivation. Together the deactivated fields and areas 1 and 2 form part of a network for reaching and grasping in human and nonhuman primates. Cooling area 7b had a dramatic effect on receptive field size for neurons in areas 1 and 2, while cooling area 5 had moderate effects and cooling M1/PM had little effect. Specifically, cooling discrete locations in 7b resulted in expansions of the receptive fields for neurons in areas 1 and 2 that were greater in magnitude and occurred in a higher proportion of sites than similar changes evoked by cooling the other fields. At some sites, the neural receptive field returned to the precooling configuration within 5-22 min of rewarming, but at other sites changes in receptive fields persisted. These results indicate that there are profound top-down influences on sensory processing of early cortical areas in the somatosensory cortex.
Assuntos
Mãos/fisiologia , Neurônios/fisiologia , Lobo Parietal/fisiologia , Percepção do Tato/fisiologia , Animais , Temperatura Baixa , Feminino , Macaca mulatta , Masculino , Microeletrodos , Atividade Motora/fisiologia , Córtex Motor/fisiologia , Estimulação FísicaRESUMO
Here we present facile microfabrication processes, referred to as Print-to-Pattern dry film photoresist (DFP) lithography, that utilize the combined advantages of wax printing and DFP to produce micropatterned substrates with high resolution over a large surface area in a non-cleanroom setting. The Print-to-Pattern methods can be performed in an out-of-cleanroom environment making microfabrication much more accessible to minimally equipped laboratories. Two different approaches employing either wax photomasks or wax etchmasks from a solid ink desktop printer have been demonstrated that allow the DFP to be processed in a negative tone or positive tone fashion, respectively, with resolutions of 100 µm. The effect of wax melting on resolution and as a bonding material was also characterized. In addition, solid ink printers have the capacity to pattern large areas with high resolution which was demonstrated by stacking DFP layers in a 50 mm × 50 mm woven pattern with 1 mm features. By using an office printer to generate the masking patterns, the mask designs can be easily altered in a graphic user interface to enable rapid prototyping.
RESUMO
Droplet microfluidics-based single-cell encapsulation is a critical technology that enables large-scale parallel single-cell analysis by capturing and processing thousands of individual cells. As the efficiency of passive single-cell encapsulation is limited by Poisson distribution, active single-cell encapsulation has been developed to theoretically ensure that each droplet contains one cell. However, existing active single-cell encapsulation technologies still face issues related to fluorescence labeling and low throughput. Here, we present an active single-cell encapsulation technique by using microvalve-based drop-on-demand technology and real-time image processing to encapsulate single cells with high throughput in a label-free manner. Our experiments demonstrated that the single-cell encapsulation system can encapsulate individual polystyrene beads with 96.3 % efficiency and HeLa cells with 94.9 % efficiency. The flow speed of cells in this system can reach 150 mm/s, resulting in a corresponding theoretical encapsulation throughput of 150 Hz. This technology has significant potential in various biomedical applications, including single-cell omics, secretion detection, and drug screening.
Assuntos
Análise de Célula Única , Humanos , Análise de Célula Única/métodos , Células HeLa , Processamento de Imagem Assistida por Computador , Poliestirenos/química , Técnicas Analíticas Microfluídicas/instrumentação , Dispositivos Lab-On-A-Chip , Encapsulamento de Células/métodosRESUMO
In recent years, micropatterning techniques have gained increasing popularity from a broad range of engineering and biology communities for the promise to establish highly quantitative investigations on miniature biological objects (e.g., cells and bacteria) with spatially defined microenvironments. However, majority of the existing techniques rely on cleanroom-based microfabrication and cannot be easily extended to a regular biological laboratory. In this paper, we present a simple versatile printing-based method, referred to as Print-to-Print (P2P), to form multi-object micropatterns for potential biological applications, along with our recent efforts to deliver out-of-cleanroom microfabrication solutions to the general public (Zhao et al. 2009), (Xing et al. 2011), (Wang et al. 2009), (Pan and Wang 2011), (Zhao et al. 2011). The P2P method employs only a commercially available solid-phase printer and custom-made superhydrophobic films. The entire patterning process does not involve any thermal or chemical treatment. Moreover, the non-contact nature of droplet transferring and printing steps can be highly advantageous for sensitive biological uses. Using the P2P process, a minimal feature resolution of 229 ± 17 µm has been successfully demonstrated. In addition, this approach has been applied to form biological micropatterning on various substrates as well as multi-object co-patterns on the commonly used surfaces. Finally, the reusability of superhydrophobic substrates has also been illustrated.
Assuntos
Membranas Artificiais , Impressão Molecular/instrumentação , Impressão Molecular/métodos , Nanopartículas/química , Nanopartículas/ultraestrutura , Nanotecnologia/instrumentação , Nanotecnologia/métodos , Desenho de Equipamento , Análise de Falha de Equipamento , Propriedades de SuperfícieRESUMO
Glaucoma is a progressive optic neuropathy in the eye, which is a leading cause of irreversible blindness worldwide and currently affects over 70 million individuals. Clinically, intraocular pressure (IOP) reduction is the only proven treatment to halt the progression of glaucoma. Microfluidic devices such as glaucoma drainage devices (GDDs) and minimally invasive glaucoma surgery (MIGS) devices are routinely used by ophthalmologists to manage elevated IOP, by creating an artificial pathway for the over-accumulated aqueous humor (AH) in a glaucomatous eye, when the natural pathways are severely blocked. Herein, a detailed modelling and analysis of both the natural microfluidic pathways of the AH in the eye and artificial microfluidic pathways formed additionally by the various glaucoma implants are conducted to provide an insight into the causes of the IOP abnormality and the improvement schemes of current implant designs. The mechanisms of representative glaucoma implants have been critically reviewed from the perspective of microfluidics, and we have categorized the current implants into four groups according to the targeted drainage sites of the AH, namely Schlemm's canal, suprachoroidal space, subconjunctival space, and ocular surface. In addition, we propose to divide the development and evolution of glaucoma implant designs into three technological waves, which include microtube (1st), microvalve (2nd) and microsystem (3rd). With the emerging trends of minimal invasiveness and artificial intelligence in the development of medical implants, we envision that a comprehensive glaucoma treatment microsystem is on the horizon, which is featured with active and wireless control of IOP, real-time continuous monitoring of IOP and aqueous rate, etc. The current review could potentially cast light on the unmatched needs, challenges, and future directions of the microfluidic structural and functional designs of glaucoma implants, which would enable an enhanced safety profile, reduced complications, increased efficacy of lowering IOP and reduced IOP fluctuations, closed-loop and on-demand control of IOP, etc.
Assuntos
Implantes para Drenagem de Glaucoma , Glaucoma , Humanos , Microfluídica , Inteligência Artificial , Glaucoma/cirurgiaRESUMO
Fluorescence imaging flow cytometry (IFC) has been demonstrated as a crucial biomedical technique for analyzing specific cell subpopulations from heterogeneous cellular populations. However, the high-speed flow of fluorescent cells leads to motion blur in cell images, making it challenging to identify cell types from the raw images. In this study, we present a real-time single-cell imaging and classification system based on a fluorescence microscope and deep learning algorithm, which is able to directly identify cell types from motion-blur images. To obtain annotated datasets of blurred images for deep learning model training, we developed a motion deblurring algorithm for the reconstruction of blur-free images. To demonstrate the ability of this system, deblurred images of HeLa cells with various fluorescent labels and HeLa cells at different cell cycle stages were acquired. The trained ResNet achieved a high accuracy of 96.6% for single-cell classification of HeLa cells in three different mitotic stages, with a short processing time of only 2 ms. This technology provides a simple way to realize single-cell fluorescence IFC and real-time cell classification, offering significant potential in various biological and medical applications.