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1.
Talanta ; 233: 122589, 2021 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-34215079

RESUMO

Digital bioassays are powerful methods to detect rare analytes from complex mixtures and study the temporal processes of individual entities within biological systems. In digital bioassays, a crucial first step is the discretization of samples into a large number of identical independent partitions. Here, we developed a rapid and facile sample partitioning method for versatile digital bioassays. This method is based on a detachable self-digitization (DSD) chip which couples a reversible assembly configuration and a predegassing-based self-pumping mechanism to achieve an easy, fast, and large-scale sample partitioning. The DSD chip consists of a channel layer used for loading the sample and a microwell layer used for holding the sample partitions. Benefitting from its detachability, the chip avoids a lengthy oil flushing process used to remove the excess sample in loading channels and can compartmentalize a sample into more than 100,000 wells of picoliter volume with densities up to 14,000 wells/cm2 in less than 30 s. We also demonstrated the utility of the proposed method by applying it to digital PCR and digital microbial assays.


Assuntos
Bioensaio , Análise de Sequência com Séries de Oligonucleotídeos , Reação em Cadeia da Polimerase
2.
Talanta ; 233: 122407, 2021 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-34215097

RESUMO

Recent virus outbreaks have revealed a critical need for large scale serological assays. However, many available tests either require a cumbersome, costly apparatus or lack the availability of full automation. In order to address these limitations, we describe a homogeneous assay for antibody detection via measurement of superparamagnetic particles agglutination. Application of a magnetic field permits to overcome the limitations governed by Brownian translational diffusion in conventional assays and results in an important acceleration of the aggregation process as well as an improvement of the limit of detection. Furthermore, the use of protein-concentrated fluid such as 5 times-diluted human plasma does not impair the performances of the method. Screening of human plasma samples shows a strict discrimination between seropositive and seronegative samples in an assay duration as short as 14 s. The sensitivity of this method, combined with its quickness and simplicity, makes it a promising diagnostic tool.


Assuntos
Aglutinação , Bioensaio , Humanos , Imunoensaio , Campos Magnéticos , Programas de Rastreamento , Sensibilidade e Especificidade
3.
Talanta ; 233: 122503, 2021 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-34215119

RESUMO

Brevetoxins (BTX) are pharmacologically active, lipid soluble cyclic polyether neurotoxins that are known to cause a wide range of neurological symptoms in humans.Harvesting and consumption of infected molluscs provide an entry point for BTXs into, the food chain, causing long-term health effects on accumulation for individuals, commonly in people with a compromised immune system and existing allergies. This study is an acoustic assay that has been constructed using a 9 MHz AT-cut quartz crystal resonator modified by attaching a specific single-stranded DNA aptamer. The DNA oligo modifies its conformation to attach itself to the binding site of the incoming BTX molecule resulting in a change in frequency on the QCR. A small Δf value was observed for lower concentrations of BTX indicating a small change in mass deposited on the crystal surface, while the opposite was true for higher concentrations. Cross-species behavior was evaluated using samples of similar origin, molecular weight and a combination of two toxins. The LOD of the fabricated QCR is 220 nM which is lower than the maximum recommended residue limit in food samples. Fresh mussel samples were spiked with known concentrations of BTX to evaluate its sensitivity in a food matrix. No interaction with other compounds was observed. Overall, this sensor finds potential application in the food sector (fishing units) where mussels are tested and graded for allergens and toxins before reaching the customer.


Assuntos
Bivalves , Oxocinas , Animais , Bioensaio , Humanos , Toxinas Marinhas , Alimentos Marinhos , Frutos do Mar/análise
4.
Molecules ; 26(11)2021 Jun 07.
Artigo em Inglês | MEDLINE | ID: mdl-34200415

RESUMO

Homovanillic acid (HVA) and vanillylmandelic acid (VMA) are end-stage metabolites of catecholamine and are clinical biomarkers for the diagnosis of neuroblastoma. For the first time in Korea, we implemented and validated a liquid chromatography tandem mass spectrometry (LC-MS/MS) assay to measure urinary concentrations of HVA and VMA according to Clinical and Laboratory Standards Institute guidelines. Our LC-MS/MS assay with minimal sample preparation was validated for linearity, lower limit of detection (LOD), lower limit of quantification (LLOQ), precision, accuracy, extraction recovery, carryover, matrix effect, and method comparison. A total of 1209 measurements was performed to measure HVA and VMA in spot urine between October 2019 and September 2020. The relationship between the two urinary markers, HVA and VMA, was analyzed and exhibited high agreement (89.1% agreement, kappa's k = 0.6) and a strong correlation (Pearson's r = 0.73). To our knowledge, this is the first study to utilize LC-MS/MS for simultaneous quantitation of spot urinary HVA and VMA and analyze the clinical application of both markers on a large scale for neuroblastoma patients.


Assuntos
Ácido Homovanílico/química , Neuroblastoma/diagnóstico , Neuroblastoma/metabolismo , Ácido Vanilmandélico/química , Bioensaio/métodos , Biomarcadores/metabolismo , Criança , Pré-Escolar , Cromatografia Líquida/métodos , Feminino , Seguimentos , Humanos , Lactente , Recém-Nascido , Limite de Detecção , Masculino , República da Coreia , Espectrometria de Massas em Tandem/métodos
5.
Molecules ; 26(11)2021 May 21.
Artigo em Inglês | MEDLINE | ID: mdl-34063887

RESUMO

The present work describes the use of Centrifugal Partition Chromatography (CPC) for the bio-guided isolation of repellent active volatile compounds from essential oils. Five essential oils (EOs) obtained from three Pinus and two Juniperus species were initially analyzed by gas chromatography-mass spectrometry (GC/MS) and evaluated for their repellent properties against Aedes albopictus. The essential oil from needles of P. pinea (PPI) presented the higher activity, showing 82.4% repellency at a dose of 0.2 µL/cm2. The above EO, together with the EO from the fruits of J. oxycedrus subsp. deltoides (JOX), were further analyzed by CPC using the biphasic system n-Heptane/ACN/BuOH in ratio 1.6/1.6/0.2 (v/v/v). The analysis of PPI essential oil resulted in the recovery of (-)-limonene, guaiol and simple mixtures of (-)-limonene/ß-pheladrene, while the fractionation of JOX EO led to the recovery of ß-myrcene, germacrene-D, and mixtures of α-pinene/ß-pinene (ratio 70/30) and α-pinene/germacrene D (ratio 65/45). All isolated compounds and recovered mixtures were tested for their repellent activity. From them, (-)-limonene, guaiol, germacrene-D as well the mixtures of (-)-limonene/ß-pheladrene presented significant repellent activity (>97% repellency) against Ae. albopictus. The present methodology could be a valuable tool in the effort to develop potent mosquito repellents which are environmentally friendly.


Assuntos
Aedes/efeitos dos fármacos , Cromatografia/métodos , Repelentes de Insetos/isolamento & purificação , Animais , Bioensaio , Cromatografia Gasosa-Espectrometria de Massas , Humanos , Repelentes de Insetos/administração & dosagem , Repelentes de Insetos/farmacologia , Juniperus/química , Óleos Voláteis/química , Pinus/química , Volatilização
6.
Nat Commun ; 12(1): 3380, 2021 06 07.
Artigo em Inglês | MEDLINE | ID: mdl-34099714

RESUMO

Plant-parasitic nematodes (PPNs) are economically important pests of agricultural crops, and soybean cyst nematode (SCN) in particular is responsible for a large amount of damage to soybean. The need for new solutions for controlling SCN is becoming increasingly urgent, due to the slow decline in effectiveness of the widely used native soybean resistance derived from genetic line PI 88788. Thus, developing transgenic traits for controlling SCN is of great interest. Here, we report a Bacillus thuringiensis delta-endotoxin, Cry14Ab, that controls SCN in transgenic soybean. Experiments in C. elegans suggest the mechanism by which the protein controls nematodes involves damaging the intestine, similar to the mechanism of Cry proteins used to control insects. Plants expressing Cry14Ab show a significant reduction in cyst numbers compared to control plants 30 days after infestation. Field trials also show a reduction in SCN egg counts compared with control plants, demonstrating that this protein has excellent potential to control PPNs in soybean.


Assuntos
Toxinas de Bacillus thuringiensis/genética , Produtos Agrícolas/parasitologia , Resistência à Doença/genética , Endotoxinas/genética , Proteínas Hemolisinas/genética , Soja/parasitologia , Tylenchoidea/patogenicidade , Animais , Bacillus thuringiensis/genética , Toxinas de Bacillus thuringiensis/metabolismo , Bioensaio , Caenorhabditis elegans , Produtos Agrícolas/genética , Produtos Agrícolas/metabolismo , Endotoxinas/metabolismo , Feminino , Engenharia Genética , Proteínas Hemolisinas/metabolismo , Melhoramento Vegetal/métodos , Doenças das Plantas/genética , Doenças das Plantas/parasitologia , Plantas Geneticamente Modificadas/genética , Plantas Geneticamente Modificadas/metabolismo , Plantas Geneticamente Modificadas/parasitologia , Soja/genética , Soja/metabolismo , Tylenchoidea/isolamento & purificação
7.
Methods Mol Biol ; 2276: 143-151, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34060038

RESUMO

Deoxynucleoside 5'-triphosphates (dNTPs) are the molecular building blocks for DNA synthesis, and their balanced concentration in the cell is fundamental for health. dNTP imbalance can lead to genomic instability and other metabolic disturbances, resulting in devastating mitochondrial diseases.The accurate and efficient measurement of dNTPs from different biological samples and cellular compartments is vital to understand the mechanisms behind these diseases and develop and scrutinize their possible treatments. This chapter describes an update on the most recent development of the traditional radiolabeled polymerase extension method and its adaptation for the measurement of whole-cell and mitochondrial dNTP pools from cultured cells and tissue samples. The solid-phase reaction setting enables an increase in efficiency, accuracy, and measurement scale.


Assuntos
Bioensaio/métodos , Fracionamento Celular/métodos , Células/metabolismo , Desoxirribonucleotídeos/metabolismo , Mitocôndrias/metabolismo , Animais , Células Cultivadas , DNA Mitocondrial/genética , DNA Mitocondrial/metabolismo , Humanos , Camundongos , Mitocôndrias/genética
8.
Methods Mol Biol ; 2276: 409-423, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34060058

RESUMO

Platinum-based antitumor drugs play important roles in the clinical treatment of various tumors. Nevertheless, some deficiencies such as poor targeting ability, low bioavailability, in vivo deactivation, drug resistance, and side effects undermine the efficacy of these drugs. Mitochondria are important organelles which regulate the energy metabolism, physiological function, life span, and survival of the cells. Regulating or interfering with mitochondrial metabolism is of great significance in the prevention or treatment of cancers. Thus, a series of mitochondrion-targeted platinum complexes were prepared by modifying triphenylphosphine (TPP+) through chemical modifications, which endow traditional platinum drugs with new properties and mechanisms through interfering with mitochondrial DNA (mtDNA), mitochondrial membrane potential (MMP), mitochondrial morphology, mitochondrial bioenergetics, or production of reactive oxygen species (ROS), thereby opening a new path for the clinical application of platinum drugs. Here we introduce the synthesis of some TPP+-modified platinum (II, IV) complexes in details and the detection method of the activity parameters related to the mitochondrial functions.


Assuntos
Bioensaio/métodos , Mitocôndrias/metabolismo , Neoplasias/tratamento farmacológico , Compostos Organofosforados/química , Compostos Organoplatínicos/farmacologia , Antineoplásicos/farmacologia , Apoptose , Linhagem Celular Tumoral , Metabolismo Energético , Humanos , Potencial da Membrana Mitocondrial , Mitocôndrias/efeitos dos fármacos , Neoplasias/química , Neoplasias/metabolismo , Espécies Reativas de Oxigênio/metabolismo
9.
Methods Mol Biol ; 2268: 119-136, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34085265

RESUMO

During the past decade, fluorescence methods have become valuable tools for characterizing ligand binding to G protein-coupled receptors (GPCRs). However, only a few of the assays enable studying wild-type receptors and monitor the ligand binding in real time. One of the approaches that is inherently suitable for this purpose is the fluorescence anisotropy (FA) assay. In the FA assay, the change of ligand's rotational freedom connected with its binding to the receptor can be monitored with a conventional fluorescence plate reader equipped with suitable optical filters. To achieve the high receptor concentration required for the assay and the low autofluorescence levels essential for reliable results, budded baculoviruses that display GPCRs on their surfaces can be used. The monitoring process generates a substantial amount of kinetic data, which is usually stored as a proprietary file format limiting the flexibility of data analysis. To solve this problem, we propose the use of the data curation software Aparecium ( http://gpcr.ut.ee/aparecium.html ), which integrates experimental data with metadata in a Minimum Information for Data Analysis in Systems Biology (MIDAS) format. Aparecium enables data export to different software packages for fitting to suitable kinetic or equilibrium models. A combination of the FA assay with the novel data analysis strategy is suitable for screening new active compounds, but also for modeling complex systems of ligand binding to GPCRs. We present the proposed approach using different fluorescent probes and assay types to characterize ligand binding to melanocortin 4 (MC4) receptor.


Assuntos
Baculoviridae/genética , Carbocianinas/química , Polarização de Fluorescência/métodos , Corantes Fluorescentes/química , Receptor Tipo 4 de Melanocortina/metabolismo , Proteínas Recombinantes/metabolismo , Animais , Ligação Competitiva , Bioensaio/métodos , Humanos , Cinética , Ligantes , Ligação Proteica , Receptor Tipo 4 de Melanocortina/química , Receptor Tipo 4 de Melanocortina/genética , Células Sf9
10.
Methods Mol Biol ; 2268: 149-157, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34085267

RESUMO

G protein-coupled receptors (GPCR) are one of the principal class of membrane proteins and around 30% of the currently marketed drugs act on one of them. The efficacious detection of ligands with the desired pharmacological profile remains a challenge of paramount importance in the GPCR drug discovery and pharmacological research. Recent evidences demonstrate that GPCR ligands can stabilize distinct receptor conformation and trigger various signaling pathways with different efficacies and/or potencies. This phenomenon called functional selectivity or biased signaling may lead to improved drugs with fewer side effects. Most receptors are promiscuous and can couple to more than one G protein family. To enable the discovery of biased ligands able to selectively trigger one G protein pathway over another, simple and efficient screening procedures are needed. The traditional assays aiming at detecting G protein activation monitor the generation of second messengers ([Ca2+]i, cAMP, IP1) or active G proteins (with GTP-g-S for instance). While these approaches have proven sensitive and robust, they are not suited for the detection of a single GPCR-G protein interaction. Here, we present in detail a method to assess directly the interaction between the receptor and the G protein. It permits the profiling of a receptor or a ligand toward G protein interactions and is compatible with high-throughput screening.


Assuntos
Bioensaio/métodos , Descoberta de Drogas/métodos , Proteínas de Ligação ao GTP/metabolismo , Luciferases/metabolismo , Nanotecnologia/métodos , Receptores Acoplados a Proteínas G/metabolismo , Células HEK293 , Humanos , Ligantes , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas
11.
Methods Mol Biol ; 2268: 193-205, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34085270

RESUMO

Intracellular calcium mobilization can be measured using several methods varying in indicator dyes and devices used. In this chapter, we describe the fluorescence-based method (FLIPR Calcium 4 Assay) developed by Molecular Devices for a FlexStation and routinely used in our laboratory for detecting intracellular calcium changes. The assay is designed to study calcium mobilization induced by majority of GPCRs and calcium channels and allows for simultaneous concentration-dependent analysis of several receptor agonists and antagonists, useful in receptor characterization and drug discovery projects.


Assuntos
Bioensaio/métodos , Canais de Cálcio/metabolismo , Cálcio/metabolismo , Descoberta de Drogas/métodos , Fluorometria/métodos , Ensaios de Triagem em Larga Escala/métodos , Receptores Acoplados a Proteínas G/metabolismo , Células Cultivadas , Humanos , Transdução de Sinais
12.
Methods Mol Biol ; 2268: 207-221, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34085271

RESUMO

GPCRs are responsible for activation of numerous downstream effectors. Live cell imaging of these effectors therefore provides a real-time readout of GPCR activity and allows for better understanding of temporal dynamics of GPCR-mediated signaling. Opsins, or optically activatable GPCRs, allow for these signaling pathways to be activated in a spatiotemporally precise and reversible manner. Here, we describe optogenetic methods for activating Gi, Gq, and Gs signaling pathways. Additionally, we present assays for detecting activation of these pathways in real time through live cell imaging of Gßγ translocation, PIP3 increase, PIP2 hydrolysis, cAMP production, and cell migration. These assays can be utilized for GPCR-targeted drug development, as well as for studies of a wide range of GPCR-mediated physiological processes.


Assuntos
Bioensaio/métodos , Transferência Ressonante de Energia de Fluorescência/métodos , Imagem Molecular/métodos , Opsinas/metabolismo , Optogenética/métodos , Receptores Acoplados a Proteínas G/metabolismo , Análise de Célula Única/métodos , Movimento Celular/fisiologia , Células Cultivadas , Humanos , Opsinas/genética , Receptores Acoplados a Proteínas G/genética , Transdução de Sinais
13.
Methods Mol Biol ; 2268: 223-232, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34085272

RESUMO

Split-TEV assay enables the identification of protein-protein interaction in mammalian cells. This method is based on the split of tobacco etch virus (TEV) protease in two fragments, where each fragment is fused to the candidate proteins predicted to interact. If there is indeed an interaction between both proteins, TEV protease reconstitutes its proteolytic activity and this activity is used to induce the expression of some reporter genes. However, some studies have detected unspecific interaction between membrane proteins due to its higher tendency to aggregate. Here we describe a variation of the Split-TEV method developed with the aim to increase the specificity in the study of G protein-coupled receptor (GPCR) interacting proteins. This approach for monitoring interactions between GPCRs is an easy and robust assay and offers good perspectives in drug discovery.


Assuntos
Bioensaio/métodos , Endopeptidases/metabolismo , Proteínas de Ligação ao GTP/metabolismo , Potyvirus/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Células Cultivadas , Genes Reporter , Humanos , Imagem Molecular/métodos , Potyvirus/genética , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Receptores Acoplados a Proteínas G/genética , Transdução de Sinais , Análise de Célula Única/métodos
14.
Methods Mol Biol ; 2268: 249-274, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34085274

RESUMO

An understanding of the kinetic contributions to G protein-coupled receptor pharmacology and signaling is increasingly important in compound profiling. Nonequilibrium conditions are commonly present in vivo, for example, as the drug competes with dynamic changes in hormone or neurotransmitter concentration for the receptor. Under such conditions individual binding kinetic properties of the ligands can influence duration of action, local ligand concentration, and functional properties such as the degree of insurmountable inhibition. Mapping the kinetic patterns of GPCR signaling events elicited by agonists, rather than a peak response at a single timepoint, is often key to predicting their functional impact. This is also a path to a better understanding of the origins of ligand bias, and whether such ligands demonstrate their effects through selection of distinct GPCR conformations, or via their kinetic properties. Recent developments in complementation approaches, based on a small bright shrimp luciferase Nanoluc, provide a new route to kinetic analysis of GPCR signaling in living cells that is amenable to the throughput required for compound profiling. In the NanoBiT luciferase complementation system, GPCRs and effector proteins are tagged with Nanoluc fragments optimized for their low interacting affinity and stability. The interactions brought about by GPCR recruitment of the effector are reproduced by a rapid and reversible increase in NanoBiT luminescence, in the presence of its substrate furimazine. Here we discuss the methods for optimizing and validating the GPCR NanoBiT assays, and protocols for their application to study endpoint and kinetic aspects of agonist and antagonist pharmacology. We also describe how timecourse families of agonist concentration response curves, derived from a single NanoBiT assay experiment, can be used to evaluate the kinetic components in operational model derived parameters of ligand bias.


Assuntos
Bioensaio/métodos , Luciferases/metabolismo , Imagem Molecular/métodos , Receptores Acoplados a Proteínas G/metabolismo , Análise de Célula Única/métodos , Células HEK293 , Humanos , Cinética , Ligantes , Luminescência , Transdução de Sinais
15.
Rev Sci Tech ; 40(1): 217-226, 2021 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-34140729

RESUMO

Any modification to a validated assay must be evaluated in terms of the impact on the assay's performance characteristics and whether the assay remains fit for the intended purpose. The comparison is referred to as a 'method comparison', 'method comparability', 'method change', or 'comparative validation'. This review presents recommendations and examples of studies found in the current literature as a means of assessing minor modifications. In addition, the authors discuss common statistical approaches used for these comparisons.


Assuntos
Bioensaio
16.
Rev Sci Tech ; 40(1): 205-215, 2021 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-34140730

RESUMO

A reliable laboratory assay is an essential tool for the diagnosis or surveillance of most animal diseases. Before routine use, assays should be appropriately validated to ensure that they have performance characteristics that provide reliable results and can be used for the intended purpose. It is inevitable that, over time, changes will need to be made to assay reagents, to the assay format, to test a different species or for implementation in a new laboratory. Whenever there is a change (whether it be components, application or location), it is essential to establish whether the new circumstances affect the biological basis and properties of the assay. If the modifications do not affect the biological basis of the assay, the changes might be considered minor and a verification study can be conducted to confirm that the performance characteristics have not been adversely affected. Major changes require a new validation to be carried out. A method comparability study, where original and modified assays are run concurrently to test the same sample panel, provides an extremely robust comparison. However, comparability studies are not always an option, especially for the introduction of a method to a new laboratory. Access to original validation data and suitable reference sample panels then becomes essential to provide evidence that the assay remains 'fit for the intended purpose'.


Assuntos
Doenças dos Animais , Doenças dos Animais/diagnóstico , Animais , Bioensaio
17.
ACS Sens ; 6(6): 2108-2124, 2021 06 25.
Artigo em Inglês | MEDLINE | ID: mdl-34076428

RESUMO

Readily deployable, low-cost point-of-care medical devices such as lateral flow assays (LFAs), microfluidic paper-based analytical devices (µPADs), and microfluidic thread-based analytical devices (µTADs) are urgently needed in resource-poor settings. Governed by the ASSURED criteria (affordable, sensitive, specific, user-friendly, rapid and robust, equipment-free, and deliverability) set by the World Health Organization, these reliable platforms can screen a myriad of chemical and biological analytes including viruses, bacteria, proteins, electrolytes, and narcotics. The Ebola epidemic in 2014 and the ongoing pandemic of SARS-CoV-2 have exemplified the ever-increasing importance of timely diagnostics to limit the spread of diseases. This review provides a comprehensive survey of LFAs, µPADs, and µTADs that can be deployed in resource-limited settings. The subsequent commercialization of these technologies will benefit the public health, especially in areas where access to healthcare is limited.


Assuntos
COVID-19 , Sistemas Automatizados de Assistência Junto ao Leito , Bioensaio , Humanos , Dispositivos Lab-On-A-Chip , SARS-CoV-2
18.
Behav Processes ; 189: 104439, 2021 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-34087348

RESUMO

In the present study we analysed spatial learning in Vespula germanica wasps when dealing with a walking Y-maze. We recorded the time taken to leave the maze during two consecutive visits and which of the two short arms was chosen to exit. Two treatments were conducted to evaluate whether wasps learned to leave the Y-maze guided either by spatial or visual cues. In Treatment 1, the colour of both arms remained unchanged between two consecutive visits; and in Treatment 2, the position of the coloured arm was switched after the first trial. Our results demonstrated that the time taken to exit the maze on the second trial was less than half in both treatments and wasps left the maze from the previously chosen arm, irrespective of its colour. This is the first study to demonstrate spatial learning in V. germanica wasps by using a walking Y-maze. Free flying wasps learned to enter the Y-maze on their own volition, walk through it, collect food and find their way out more rapidly after a single foraging experience. The current experimental device is suitable for the evaluation of spatial memory processes and exploratory behaviour in this species.


Assuntos
Vespas , Animais , Bioensaio , Sinais (Psicologia) , Aprendizagem em Labirinto , Projetos Piloto , Memória Espacial
20.
Anal Chem ; 93(24): 8622-8630, 2021 06 22.
Artigo em Inglês | MEDLINE | ID: mdl-34110770

RESUMO

Water-in-oil emulsion droplet microfluidic systems have been extensively developed, and currently, almost all cell handling steps can be conducted in this format. An exception is the cell washing and solution exchange step, which is commonly utilized in many conventional cell assays. This paper presents an in-droplet cell washing and solution exchange technology that utilizes dielectrophoretic (DEP) force to move all cells to one side of a droplet, followed by asymmetrical splitting of the droplet to obtain a small daughter droplet that contains all or most of the cells, and then finally merges this cell-concentrated droplet with a new droplet that contains the desired solution. These sequential droplet manipulation steps were integrated into a single platform, where up to 88% of the original solution in the droplet could be exchanged with the new solution while keeping cell loss to less than 5%. Two application examples were demonstrated using the developed technology. In the first example, green microalga Chlamydomonas reinhardtii cells were manipulated using negative DEP force to exchange the regular culture medium with a nitrogen-limited medium to induce lipid production. In the second example, Salmonella enterica cells were manipulated using positive DEP force to replace fluorescent dye that models fluorescent cell stains that contribute to high background noise in fluorescence-based droplet content detection with fresh buffer solution, significantly improving the droplet content detection sensitivity. Since the cell washing step is one of the most frequently utilized steps in many cell biology assays, we expect that the developed technology can significantly broaden the type of assay that can be conducted in droplet microfluidic format.


Assuntos
Chlamydomonas reinhardtii , Técnicas Analíticas Microfluídicas , Bioensaio , Emulsões , Microfluídica
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