RESUMO
SignificanceMany microbial populations proliferate in small channels. In such environments, reproducing cells organize in parallel lanes. Reproducing cells shift these lanes, potentially expelling other cells from the channel. In this paper, we combine theory and experiments to understand how these dynamics affects the diversity of a microbial population. We theoretically predict that genetic diversity is quickly lost along lanes of cells. Our experiments confirm that a population of proliferating Escherichia coli in a microchannel organizes into lanes of genetically identical cells within a few generations. Our findings elucidate the effect of lane formation on populations evolution, with potential applications ranging from microbial ecology in soil to dynamics of epithelial tissues in higher organisms.
Assuntos
Escherichia coli , Genética Populacional , Escherichia coli/genética , SoloRESUMO
In nature, chemotactic interactions are ubiquitous and play a critical role in driving the collective behavior of living organisms. Reproducing these interactions in vitro is still a paramount challenge due to the complexity of mimicking and controlling cellular features, such as tangled metabolic networks, cytosolic macromolecular crowding, and cellular migration, on a microorganism size scale. Here, we generate enzymatically active cell-sized droplets able to move freely, and by following a chemical gradient, able to interact with the surrounding droplets in a collective manner. The enzyme within the droplets generates a pH gradient that extends outside the edge of the droplets. We discovered that the external pH gradient triggers droplet migration and controls its directionality, which is selectively toward the neighboring droplets. Hence, by changing the enzyme activity inside the droplet, we tuned the droplet migration speed. Furthermore, we showed that these cellular-like features can facilitate the reconstitution of a simple and linear protometabolic pathway and increase the final reaction product generation. Our work suggests that simple and stable membraneless droplets can reproduce complex biological phenomena, opening new perspectives as bioinspired materials and synthetic biology tools.
Assuntos
Quimiotaxia , Concentração de Íons de Hidrogênio , Tamanho da PartículaRESUMO
The gene therapy field has advanced in recent years with five recombinant adeno-associated virus (rAAV) based products winning Food and Drug Administration (FDA) approval. As the number of therapeutic applications and overall production demands for rAAV increase, it is valuable to evaluate rAAV production in different production cells. Chinese hamster ovary (CHO) cells have been a robust host for biomolecule manufacturing for more than 35 years. However, there is no report to our knowledge describing the use of CHO cells for rAAV production. In this study, we examined the ability of CHO cells to produce rAAV using a transient plasmid transfection approach. Our results demonstrated that CHO is capable of producing rAAV with detectable viral fundamental components including viral RNAs, proteins, and rAAV viral particles. We identified the expression of cap proteins as one of the limiting factors for rAAV production in CHO cells. We therefore added an additional cytomegalovirus (CMV)-Cap plasmid to the CHO transfection. After increasing cap protein expression, we detected rAAV titers as high as 3 × 108 viral genomes for every 2 × 109 capsids in CHO cells using a quintuple transfection method (standard AAV2 Rep/Cap, helper, gene of interest plasmids, plus CMV-E1, and CMV-Cap plasmids) with comparable full particle percent (average 15%) to that of human embryo kidney (HEK)-derived rAAV. Our study provides a foundation for potential rAAV production in CHO cells.
Assuntos
Infecções por Citomegalovirus , Vetores Genéticos , Animais , Cricetinae , Humanos , Cricetulus , Células CHO , Dependovirus/genética , Plasmídeos/genéticaRESUMO
In this Feature Article, we discuss the interplay between fluidics and the localized surface plasmon resonance (LSPR) sensing technique, primarily focusing on its applications in the realm of bio/chemical sensing within fluidic environments. Commencing with a foundational overview of LSPR principles from a sensing perspective, we subsequently showcase the development of a streamlined LSPR chip integrated with microfluidic structures. This integration opens the doors to advanced experiments involving fluid dynamics, greatly expanding the scope of LSPR-based research. Our discussions then turn to the practical implementation of LSPR and microfluidics in real-time biosensing, with a specific emphasis on monitoring DNA polymerase activity. Additionally, we illustrate the direct sensing of biological fluids, exemplified by the analysis of urine, while also shedding light on a unique particle assembly process that occurs on LSPR chips. We not only discuss the significance of LSPR sensing but also explore its potential to investigate a plethora of phenomena at liquid-liquid and solid-liquid interfaces. This is particularly noteworthy, as existing transduction methods and sensors fall short in fully comprehending these interfacial phenomena. Concluding our discussion, we present a futuristic perspective that provides insights into potential opportunities emerging at the intersection of fluidics and LSPR sensing.
RESUMO
Viscoelastic properties of soft substrates play a crucial role in the evaporation dynamics of sessile drops. Recent studies have revealed that the modification of the viscoelastic properties of substrates changes the dynamics of the three-phase contact line, consequently affecting the evaporation behavior of sessile drops. Notably, these modifications occur without any noticeable changes to the substrate's wetting characteristics or surface topography. However, the individual role of storage (G') and loss (Gâ³) moduli of substrates on drop evaporation dynamics remains unexplored. In this study, we investigate the evaporation dynamics of water drops on two groups of poly(dimethylsiloxane)-based viscoelastic substrates possessing either identical G' with varying Gâ³ or identical Gâ³ with varying G'. Our study reveals that on a substrate with constant shear modulus (G'), a reduction of an order of magnitude in loss modulus shifts the evaporation process from the constant contact radius mode to the constant contact angle mode. We hypothesize that this observed shift in behavior stems from the varying viscoelastic dissipation influenced by the plateau modulus and characteristic relaxation time of polymer gels. Our hypothesis is further supported from the observation that the evaporation process persists on the substrate with constant loss modulus (Gâ³). Our study advances the current understanding of drop evaporation on soft substrates that may find potential applications involving soft composites, biological entities, tissue engineering, and wearable electronics.
RESUMO
Polyelectrolytes are a class of polymers possessing ionic groups on their repeating units. Since counterions can dissociate from the polymer backbone, polyelectrolyte chains are strongly influenced by electrostatic interactions. As a result, the physical properties of polyelectrolyte solutions are significantly different from those of electrically neutral polymers. The aim of this article is to highlight key results and some outstanding questions in the polyelectrolyte research from recent literature. We focus on the influence of electrostatics on conformational and hydrodynamic properties of polyelectrolyte chains. A compilation of experimental results from the literature reveals significant disparities with theoretical predictions. We also discuss a new class of polyelectrolytes called poly(ionic liquid)s that exhibit unique physical properties in comparison to ordinary polyelectrolytes. We conclude this review by listing some key research challenges in order to fully understand the conformation and dynamics of polyelectrolytes in solutions.
RESUMO
We present a comprehensive investigation combining numerical simulations with experimental validation, focusing on the creeping flow behavior of a shear-banding, viscoelastic wormlike micellar (WLM) solution over concavities with various depths (D) and lengths (L). The fluid is modeled using the diffusive Giesekus model, with model parameters set to quantitatively describe the shear rheology of a 100 : 60 mM cetylpyridinium chloride:sodium salicylate aqueous WLM solution used for the experimental validation. We observe a transition from "cavity flow" to "expansion-contraction flow" as the length L exceeds the sum of depth D and channel width W. This transition is manifested by a change of vortical structures within the concavity. For L ≤ D + W, "cavity flow" is characterized by large scale recirculations spanning the concavity length. For L > D + W, the recirculations observed in "expansion-contraction flow" are confined to the salient corners downstream of the expansion plane and upstream of the contraction plane. Using the numerical dataset, we construct phase diagrams in L-D at various fixed Weissenberg numbers Wi, characterizing the transitions and describing the evolution of vortical structures influenced by viscoelastic effects.
RESUMO
This study describes a numerical analysis on blue energy generation using a charged nanochannel with an integrated pH-sensitive polyelectrolyte layer (PEL), considering ion partitioning effects due to permittivity differences. The mathematical model for ionic and fluidic transport is solved using the finite element method, and the model validation is performed against existing theoretical and experimental results. The study investigates the influence of electrolyte concentration, permittivity ratio, and salt types (KCl, BeCl2, AlCl3) on the energy conversion process. The findings illustrate the substantial role of ion partitioning in modulating ionic concentration and potential fields, thereby affecting current profiles and energy conversion efficiencies. Remarkably, overlooking ion partitioning leads to significant overestimations of power density, highlighting the necessity of this consideration for accurate device performance predictions. This work introduces a promising configuration that achieves higher power densities, paving the way for the next generation of efficient energy-harvesting devices. The findings offer valuable insights into the development of state-of-the-art blue energy harvesting nanofluidic devices, advancing sustainable energy production.
RESUMO
Viscoelastic flows through porous media become unstable and chaotic beyond critical flow conditions, impacting widespread industrial and biological processes such as enhanced oil recovery and drug delivery. Understanding the influence of the pore structure or geometry on the onset of flow instability can lead to fundamental insights into these processes and, potentially, to their optimization. Recently, for viscoelastic flows through porous media modeled by arrays of microscopic posts, Walkama et al. [D. M. Walkama, N. Waisbord, J. S. Guasto, Phys. Rev. Lett 124, 164501 (2020)] demonstrated that geometric disorder greatly suppressed the strength of the chaotic fluctuations that arose as the flow rate was increased. However, in that work, disorder was only applied to one originally ordered configuration of posts. Here, we demonstrate experimentally that, given a slightly modified ordered array of posts, introducing disorder can also promote chaotic fluctuations. We provide a unifying explanation for these contrasting results by considering the effect of disorder on the occurrence of stagnation points exposed to the flow field, which depends on the nature of the originally ordered post array. This work provides a general understanding of how pore geometry affects the stability of viscoelastic porous media flows.
RESUMO
Short liquid bridges are stable under the action of surface tension. In applications like electronic packaging, food engineering, and additive manufacturing, this poses challenges to the clean and fast dispensing of viscoelastic fluids. Here, we investigate how viscoelastic liquid bridges can be destabilized by torsion. By combining high-speed imaging and numerical simulation, we show that concave surfaces of liquid bridges can localize shear, in turn localizing normal stresses and making the surface more concave. Such positive feedback creates an indent, which propagates toward the center and leads to breakup of the liquid bridge. The indent formation mechanism closely resembles edge fracture, an often undesired viscoelastic flow instability characterized by the sudden indentation of the fluid's free surface when the fluid is subjected to shear. By applying torsion, even short, capillary stable liquid bridges can be broken in the order of 1 s. This may lead to the development of dispensing protocols that reduce substrate contamination by the satellite droplets and long capillary tails formed by capillary retraction, which is the current mainstream industrial method for destabilizing viscoelastic liquid bridges.
RESUMO
The rapid transmission and resilience of coronavirus disease 2019 (COVID-19) have led to urgent demands in monitoring humoral response for effective vaccine development, thus a multiplex co-detection platform to discriminate infection-induced from vaccine-induced antibodies is needed. Here a duplex electrochemical immunosensor for co-detection of anti-nucleocapsid IgG (N-IgG) and anti-spike IgG (S-IgG) is developed by using a two-working electrode system, via an indirect immunoassay, with antibody quantification obtained by differential pulse voltammetry. The screen-printed electrodes (SPEs) are modified by carbon black and electrodeposited gold nanoflowers for maximized surface areas, enabling the construction of an immunological chain for S-IgG and N-IgG electrochemical detection with enhanced performance. Using an optimized immunoassay protocol, a wide linear range between 30-750 and 20-1000 ng mL-1 , and a limit of detection of 28 and 15 ng mL-1 are achieved to detect N-IgG and S-IgG simultaneously in serum samples. This duplex immunosensor is then integrated in a microfluidic device to obtain significantly reduced detection time (≤ 7 min) while maintaining its analytical performance. The duplex microfluidic immunosensor can be easily expanded into multiplex format to achieve high throughput screening for the sero-surveillance of COVID-19 and other infectious diseases.
Assuntos
Técnicas Biossensoriais , COVID-19 , Vacinas , Humanos , COVID-19/diagnóstico , Imunoensaio/métodos , Microfluídica , Anticorpos Antivirais , Imunoglobulina G , Técnicas Eletroquímicas/métodos , Eletrodos , OuroRESUMO
Fluids composed of biosourced rod-like colloids (RC) and rod-like polymers (RP) have been extensively studied due to various promising applications relying on their flow-induced orientation (e.g., fiber spinning). However, the relationship between RC and RP alignment and the resulting rheological properties is unclear due to experimental challenges. We investigate the alignment-rheology relationship for a variety of biosourced RC and RP, including cellulose-based particles, filamentous viruses, and xanthan gum, by simultaneous measurements of the shear viscosity and fluid anisotropy under rheometric shear flows. For each system, the RC and RP contribution to the fluid viscosity, captured by the specific viscosity ηsp, follows a universal trend with the extent of the RC and RP alignment independent of concentration. We further exploit this unique rheological-structural link to retrieve a dimensionless parameter (ß) directly proportional to ηsp at zero shear rate (η0,sp), a parameter often difficult to access from experimental rheometry for RC and RP with relatively long contour lengths. Our results highlight the unique link between the flow-induced structural and rheological changes occurring in RC and RP fluids. We envision that our findings will be relevant in building and testing microstructural constitutive models to predict the flow-induced structural and rheological evolution of fluids containing RC and RP.
Assuntos
Coloides , Polímeros , Polímeros/química , Celulose , Reologia , ViscosidadeRESUMO
Viscoelastic flows in the cross-slot geometry can undergo a transition from a steady symmetric to a steady asymmetric flow state, ostensibly due to purely-elastic effects arising beyond a critical flow rate, or Weissenberg number Wi. However, some reports suggest that shear thinning of the fluid's viscosity may also play an important role in this transition. We employ a series of polymer solutions of varying rheological properties to investigate in detail how the interplay between fluid elasticity and shear thinning affects the onset and development of asymmetric flows in the cross-slot. Flow velocimetry is performed on each of the polymer solutions, and is used to assess the degree of flow asymmetry I in the cross-slot as a function of both Wi and a dimensionless parameter S quantifying the flow-rate-dependent extent of shear thinning. Typically, the flow field breaks symmetry as Wi is increased beyond a critical value, but the magnitude of I is found to also be dependent on S. For a few specific polymer solutions, the flow field recovers symmetry above a second, higher critical Wi as S becomes small. The experimental results are summarized in a flow state diagram in Wi-S space, showing the relationship between flow asymmetry and fluid rheology. Finally, to gain a deeper understanding of the effects of shear thinning, numerical simulations are performed using the linear simplified Phan-Thien-Tanner model. We demonstrate that the degree of both shear thinning and elasticity of the fluid, and their interplay, are important factors controlling elastic instabilities in the cross-slot geometry.
RESUMO
We report experimental microfluidic measurements and theoretical modeling of elastoviscoplastic materials under steady, planar elongation. Employing a theory that allows the solid state to deform, we predict the yielding and flow dynamics of such complex materials in pure extensional flows. We find a significant deviation of the ratio of the elongational to the shear yield stress from the standard value predicted by ideal viscoplastic theory, which is attributed to the normal stresses that develop in the solid state prior to yielding. Our results show that the yield strain of the material governs the transition dynamics from the solid state to the liquid state. Finally, given the difficulties of quantifying the stress field in such materials under elongational flow conditions, we identify a simple scaling law that enables the determination of the elongational yield stress from experimentally measured velocity fields.
RESUMO
Toward the goal of increasing the throughput of high-resolution mass characterization of intact antibodies, we developed a RapidFire-mass spectrometry (MS) assay using electrospray ionization. We achieved unprecedented screening throughput as fast as 15 s/sample, which is an order of magnitude improvement over conventional liquid chromatography (LC)-MS approaches. The screening enabled intact mass determination as accurate as 7 ppm with baseline resolution at the glycoform level for intact antibodies. We utilized this assay to characterize and perform relative quantitation of antibody species from 248 samples of 62 different cell line clones at four time points in 2 h using RapidFire-time-of-flight MS screening. The screening enabled selection of clones with the highest purity of bispecific antibody production and the results significantly correlated with conventional LC-MS results. In addition, analyzing antibodies from a complex plasma sample using affinity-RapidFire-MS was also demonstrated and qualified. In summary, the platform affords high-throughput analyses of antibodies, including bispecific antibodies and potential mispaired side products, in cell culture media, or other complex matrices.
Assuntos
Anticorpos Biespecíficos/sangue , Anticorpos/sangue , Ensaios de Triagem em Larga Escala/métodos , Espectrometria de Massas por Ionização por Electrospray/métodos , Anticorpos/isolamento & purificação , Anticorpos Biespecíficos/isolamento & purificação , Linhagem Celular , Cromatografia Líquida/métodos , HumanosRESUMO
BACKGROUND: Several cases of herpes zoster-induced psoriasis have been reported in the literature. OBJECTIVE: Our nationwide retrospective cohort study is designed to examine the risk association between herpes zoster and psoriasis. METHODS: From the Taiwan National Health Insurance Research Database, 26,623 patients from 1999 to 2013 with a diagnosis of herpes zoster and no prior history of psoriasis were selected as the study subjects. The control group was established during the study period from those without a herpes zoster diagnosis and was propensity score matched to minimize confounding factors. Both cohorts were followed for cases of psoriasis development. Data analysis was done via Kaplan-Meier analysis and Cox Proportional-Hazards Models. RESULTS: Comparing the study group to control, the adjusted hazard ratio was 1.66 (95% CI, 1.31-2.13: p < 0.05) after adjusting for covariates (age, gender, urbanization, selected comorbidities and selected medications use). Statistical analysis found no interaction effect among herpes zoster and other covariates for risk modification of psoriasis development. CONCLUSION: This study demonstrated an increased risk of psoriasis in patients diagnosed with herpes zoster.
Assuntos
Herpes Zoster , Psoríase , Humanos , Estudos Retrospectivos , Estudos de Coortes , Herpes Zoster/complicações , Herpes Zoster/epidemiologia , Herpesvirus Humano 3 , Psoríase/epidemiologia , Progressão da Doença , Fatores de Risco , IncidênciaRESUMO
Residual host cell proteins (HCPs) in the drug product can affect product quality, stability, and/or safety. In particular, highly active hydrolytic enzymes at sub-ppm levels can negatively impact the shelf life of drug products but are challenging to identify by liquid chromatography-mass spectrometry/mass spectrometry (LC-MS/MS) due to their high dynamic range between HCPs and biotherapeutic proteins. We employed new strategies to address the challenge: (1) native digest at a high protein concentration; (2) sodium deoxycholate added during the reduction step to minimize the inadvertent omission of HCPs observed with native digestion; and (3) solid phase extraction with 50% MeCN elution prior to LC-MS/MS analysis to ensure effective mAb removal. A 50 cm long nanoflow charged surface hybrid column was also packed to allow for higher sample load for increased sensitivity. Our workflow has increased the sensitivity for HCP identification by 10- to 100-fold over previous reports and showed the robustness as low as 0.1 ppm for identifying HCPs (34.5 to 66.2 kDa MW). The method capability was further confirmed by consistently identifying >85% of 48 UPS-1 proteins (0.10 to 1.34 ppm, 6.3 to 82.9 kDa MW) in a monoclonal antibody (mAb) and the largest number (746) of mouse proteins from NIST mAb reported to date by a single analysis. Our work has filled a significant gap in HCP analysis for detecting and demonstrating HCP clearance, in particular, extremely low-level hydrolases in drug process development.
Assuntos
Anticorpos Monoclonais , Espectrometria de Massas em Tandem , Animais , Anticorpos Monoclonais/análise , Células CHO , Cromatografia Líquida , Cricetinae , Cricetulus , Camundongos , Espectrometria de Massas em Tandem/métodos , Fluxo de TrabalhoRESUMO
C-terminal lysine (CTK) is often classified as a potential critical quality attribute for therapeutic antibodies being developed for subcutaneous (SC) administration because of its potential to impact antibody SC bioavailability/pharmacokinetics (PK). This classification both inflates development costs and increases comparability risks for SC administration of antibodies. However, prior risk assessments of CTK have not fully considered biotransformation of CTK in the SC space, which may play an important role given that circulating carboxypeptidases in humans rapidly process CTK on intravenously administered antibodies. Here, CTK biotransformation in biofluid derived from human SC space was investigated. The representative fluid from the human SC space was sampled from 10 healthy human subjects using the suction blister method. Glycosylated antibody containing high levels of CTK (expressed using a carboxypeptidase D CRISPR/Cas9 knockout CHO cell line) was incubated in the collected suction blister fluids (SBFs), recovered using cognate antigen pulldowns, and characterized for remaining CTK levels using intact and reduced liquid chromatography-mass spectrometry (LC-MS) analysis. CTK processing (i.e., carboxypeptidase activity) was evident in all SBF and exhibited first-order kinetics with rate constants of 2.18 ± 0.57 d-1 (at 37 °C). PK simulations that integrated CTK processing pathways and their associated rate constants were subsequently performed using a range of clinically observed PK parameters for therapeutic antibodies, including atezolizumab- and pertuzumab-specific parameters. The impact of CTK content (even up to 100%) on SC PK outcomes such as bioavailability and Ctrough were modest (<14%) for all combinations of PK parameters tested in the sensitivity analysis. This study forms the cornerstone data package for derisking CTK as a PK liability for antibody SC programs and highlights the usefulness of fully considering biotransformation during product quality criticality assessments.
Assuntos
Vesícula , Lisina , Animais , Cricetinae , Humanos , Sucção , Células CHO , Cricetulus , Imunoglobulina G , Injeções SubcutâneasRESUMO
We investigate the effect of the ion diameter a of ionic liquids (ILs) on the shear viscosity of polymerized ionic liquids (PILs) in IL solutions. When both the PIL and IL contain large PFSI anions (a ≈ 0.57 nm), the specific viscosity ηsp first decreases with increasing IL concentration cIL in the low cIL regime, reaches a minimum and then increases with increasing cIL in the high cIL regime. By comparing the measured ηsp with the modified charge screening model proposed in our previous study [Matsumoto et al., Macromolecules, 2021, 54, 5648-5661], we attribute the observed non-monotonic trend of ηsp against cIL to the charge underscreening phenomenon, i.e., an increase of the screening length at high cIL leads to the upturn of ηsp. On the other hand, when the PIL and IL contain small BF4 anions (a ≈ 0.34 nm), the ηsp decreases asymptotically with increasing cIL, because the charge on the PIL chain is likely screened fully in the entire cIL regime. Our results demonstrate that the ion diameter of ILs plays an important role in governing the charge screening mechanism of PILs in IL solutions, and thus influencing the viscoelastic properties of PIL solutions.
RESUMO
We present experiments on the flow of a viscoelastic wormlike micellar solution around cylinders (radius R) confined in straight microchannels (width W). Thirteen flow geometries are tested where the blockage ratio is varied over a wide range 0.055 ≤ BR = 2R/W ≤ 0.63. Experiments are performed at negligible Reynolds number, and for Weissenberg numbers Wi = λU/R up to 1000, where U is the average flow speed and λ is the relaxation time of the fluid. Micro-particle image velocimetry is used to characterise the flow state at each BR and Wi. In all of the geometries, a first critical Weissenberg number marks a transition from symmetric flow to an asymmetric but time-steady flow state, while a second higher critical Weissenberg number marks the onset of time-dependent flows. However, we report a clear shift in behaviour over a narrow intermediate range of 0.33 â² BR â² 0.41. Channels with BR ≤ 0.33 fall in a 'low' BR regime, with instabilities that originate from the downstream stagnation point, while those with BR ≥ 0.44 fall in a 'high' BR regime, with instabilities developing at the upstream stagnation point. Behaviour within the newly-identified intermediate BR regime is complex due to the competing influence of the two stagnation points. We summarise all our results in a flow state diagram covering Wi-BR parameter space, clearly defining the different regimes of blockage ratio for the first time. Our results contribute to the understanding of the complexities of viscoelastic flow in this benchmark geometry.