Enhanced antibacterial efficacy: rapid analysis of silver-decorated azithromycin-infused Soluplus® nanoparticles against E. coli and S. epidermidis biofilms.

The escalating threat of antibiotic-resistant bacterial biofilms necessitates innovative antimicrobial strategies. This study introduces silver-decorated azithromycin-infused Soluplus® nanoparticles (Ag-AZI-Sol NPs) synthesized via a controlled emulsion diffusion method to ensure sustained release of antimicrobial silver ions for over six hours-a critical factor for continuous antibacterial efficacy. The efficacy of these nanoparticles was evaluated against biofilms formed by Escherichia coli (E. coli) and Staphylococcus epidermidis (S. epidermidis), pathogens that cause hospital-acquired infections. Concentrations of 5 and 10 µg mL-1 of Ag-AZI-Sol NPs induced significant morphological changes within the biofilms, disrupting the bacterial extracellular matrix as observed using scanning electron microscopy (SEM). This disruption peaked between two and six hours, coinciding with damage to bacterial cells by the silver ions. Antibacterial assay measurements confirmed a significant reduction in the growth rate among the Ag-AZI-Sol NP-treated bacteria compared with controls. Electrochemical analysis using laser-induced graphene (LIG) and chronoamperometry revealed a decline in current, indicating an effective antibacterial effect. This innovative biosensing technique makes use of the high conductivity and surface area of LIG to detect changes in bacterial activity quickly and sensitively. Our findings highlight the potent microbicidal properties of Ag-AZI-Sol NPs and suggest diverse applications from food processing to medical device coatings.

Wearable electrochemical device based on butterfly-like paper-based microfluidics for pH and Na+ monitoring in sweat.

A wearable potentiometric device is reported based on an innovative butterfly-like paper-based microfluidic system, allowing for continuous monitoring of pH and Na+ levels in sweat during physical activity. Specifically, the use of the butterfly-like configuration avoids evaporation phenomena and memory effects, enabling precise and timely biomarker determination in sweat. Two ad hoc modified screen-printed electrodes were embedded in the butterfly-like paper-based microfluidics, and the sensing device was further integrated with a portable and miniaturized potentiostat, leveraging Bluetooth technology for efficient data transmission. First, the paper-based microfluidic configuration was tested for optimal fluidic management to obtain optimized performance of the device. Subsequently, the two electrodes were individually tested to detect the two biomarkers, namely pH and Na+. The results demonstrated highly promising near-Nernstian (0.056 ± 0.002 V/dec) and super-Nernstian (- 0.080 ± 0.003 V/pH) responses, for Na+ and pH detection, respectively. Additionally, several important parameters such as storage stability, interferents, and memory effect by hysteresis study were also investigated. Finally, the butterfly-like paper-based microfluidic wearable device was tested for Na+ and pH monitoring during the physical activity of three volunteers engaged in different exercises, obtaining a good correlation between Na+ increase and dehydration phenomena. Furthermore, one volunteer was tested through a cardiopulmonary test, demonstrating a correlation between sodium Na+ increase and the energetic effort by the volunteer. Our wearable device highlights the high potential to enable early evaluation of dehydration and open up new opportunities in sports activity monitoring.

Flow of wormlike micellar solutions over concavities.

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.

Maximizing blue energy: the role of ion partitioning in nanochannel systems.

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.

From yield stress to elastic instabilities: Tuning the extensional behavior of elastoviscoplastic fluids.

In this study, we delve into the intricacies of elastoviscoplastic (EVP) fluids, particularly focusing on how polymer additives influence their extensional behavior. Our findings reveal that polymer additives significantly alter the extensional properties of the EVP fluids, such as relaxation time and extensional stresses while having negligible impact on the shear rheology. Interestingly, the modified fluids exhibit a transition from yield stress-like behavior to viscoelastic-like behavior under high extensional rates, ultimately leading to destabilization under extreme deformation. This research enhances the fundamental understanding of EVP fluids and highlights potential advancements in applications, especially in precision-demanding fields like 3D printing.

SPEED-MODE cell line development (CLD): Reducing Chinese hamster ovary (CHO) CLD timelines via earlier suspension adaptation and maximizing time spent in the exponential growth phase.

Chinese hamster ovary (CHO) cells are the preferred system for expression of therapeutic proteins and the majority of all biotherapeutics are being expressed by these cell lines. CHO expression systems are readily scalable, resistant to human adventitious agents, and have desirable post-translational modifications, such as glycosylation. Regardless, drug development as a whole is a very costly, complicated, and time-consuming process. Therefore, any improvements that result in reducing timelines are valuable and can provide patients with life-saving drugs earlier. Here we report an effective method (termed SPEED-MODE, herein) to speed up the Cell line Development (CLD) process in a targeted integration (TI) CHO CLD system. Our findings show that (1) earlier single cell cloning (SCC) of transfection pools, (2) speeding up initial titer screening turnaround time, (3) starting suspension adaptation of cultures sooner, and (4) maximizing the time CHO cultures spend in the exponential growth phase can reduce CLD timelines from ~4 to ~3 months. Interestingly, SPEED-MODE timelines closely match the theoretical minimum timeline for CHO CLD assuming that CHO cell division is the rate limiting factor. Clones obtained from SPEED-MODE CLD yielded comparable titer and product quality to those obtained via a standard CLD process. Hence, SPEED-MODE CLD is advantageous for manufacturing biotherapeutics in an industrial setting as it can significantly reduce CLD timelines without compromising titer or product quality.

Chemotactic Interactions Drive Migration of Membraneless Active Droplets.

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.

Understanding the Role of Loss Modulus of Viscoelastic Substrates in the Evaporation Dynamics of Sessile Drops.

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 (G″) 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 G″ or identical G″ 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 (G″). 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.

Multiplexed Opto-Microfluidic Biosensing: Advanced Platform for Prostate Cancer Detection.

Cancer stands as a prominent global cause of mortality, necessitating early detection to augment survival rates and alleviate economic burdens on healthcare systems. In particular, prostate cancer (PCa), impacting 1.41 million men globally in 2020, accentuates the demand for sensitive and cost-effective detection methods beyond traditional prostate-specific antigen (PSA) testing. While clinical techniques exhibit limitations, biosensors emerge as compact, user-friendly alternatives to traditional laboratory approaches. However, existing biosensors predominantly concentrate on PSA detection, prompting the necessity for advancing toward multiplex sensing platforms. This study introduces a compact opto-microfluidic sensor featuring a substrate of gold nanospikes, fabricated via electrodeposition, for enhanced sensitivity. Embedded within a microfluidic chip, this nanomaterial enables the precise and concurrent measurement of PSA, alongside two complementary PCa biomarkers, matrix metalloproteinase-2 (MMP-2) and anti-α-methylacyl-CoA racemase (anti-AMACR) in diluted human plasma, offering a comprehensive approach to PSA analysis. Taking advantage of the localized surface plasmon resonance principle, this biosensor offers robustness and sensitivity in real sample analysis without the need for labeling agents. With the limit of detection at 0.22, 0.37, and 0.18 ng/mL for PSA, MMP-2, and anti-AMACR, respectively, this biosensing platform holds promise for point-of-care analysis, underscoring its potential impact on medical diagnostics.

Localized Surface Plasmon Resonance Sensing and its Interplay with Fluidics.

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.

Nonclinical Investigation of Cytokine Mitigation Strategies for T-cell-Engaging Bispecifics in the Cynomolgus Macaque.

SUMMARY: T-cell-directed cancer therapies such as T-cell-engaging bispecifics (TCBs) are commonly associated with cytokine release syndrome and associated clinical signs that can limit their tolerability and therapeutic benefit. Strategies for reducing cytokine release are therefore needed. Here, we report on studies performed in cynomolgus monkeys to test different approaches for mitigating cytokine release with TCBs. A "priming dose" as well as subcutaneous dosing reduced cytokine release compared with intravenous dosing but did not affect the intended T-cell response to the bispecific. As another strategy, cytokines or cytokine responses were blocked with an anti-IL-6 antibody, dexamethasone, or a JAK1/TYK2-selective inhibitor, and the effects on toxicity as well as T-cell responses to a TCB were evaluated. The JAK1/TYK2 inhibitor and dexamethasone prevented CRS-associated clinical signs on the day of TCB administration, but the anti-IL-6 had little effect. All interventions allowed for functional T-cell responses and expected damage to target-bearing tissues, but the JAK1/TYK2 inhibitor prevented the upregulation of activation markers on T cells, suggesting the potential for suppression of T-cell responses. Our results suggest that short-term prophylactic dexamethasone treatment may be an effective option for blocking cytokine responses without affecting desired T-cell responses to TCBs.

Dilute polyelectrolyte solutions: recent progress and open questions.

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.

Gene copy number, gene configuration and LC/HC mRNA ratio impact on antibody productivity and product quality in targeted integration CHO cell lines.

The augmentation of transgene copy numbers is a prevalent approach presumed to enhance transcriptional activity and product yield. CHO cell lines engineered via targeted integration (TI) offer an advantageous platform for investigating the interplay between gene copy number, mRNA abundance, product yield, and product quality. Our investigation revealed that incrementally elevating the gene copy numbers of both IgG heavy chain (HC) and light chain (LC) concurrently resulted in the attainment of plateaus in mRNA levels and product titers, notably occurring beyond four to five gene copies integrated at the same TI site. Furthermore, maintaining a fixed gene copy number while varying the position of genes within the vector influenced the LC/HC mRNA ratio, which subsequently exerted a substantial impact on product titer. Moreover, manipulation of the LC/HC gene ratio through the introduction of surplus LC gene copies led to heightened LC mRNA expression and a reduction in the levels of high molecular weight species. It is noteworthy that the effects of excess LC on product titer were dependent on the specific molecule under consideration. The strategic utilization of PCR tags enabled precise quantification of transcription from each expression slot within the vector, facilitating the identification of highly expressive and less expressive slots. Collectively, these findings significantly enhance our understanding of stable antibody production in TI CHO cell lines.

Twisted fiber microfluidics: a cutting-edge approach to 3D spiral devices.

The development of 3D spiral microfluidics has opened new avenues for leveraging inertial focusing to analyze small fluid volumes, thereby advancing research across chemical, physical, and biological disciplines. While traditional straight microchannels rely solely on inertial lift forces, the novel spiral geometry generates Dean drag forces, eliminating the necessity for external fields in fluid manipulation. Nevertheless, fabricating 3D spiral microfluidics remains a labor-intensive and costly endeavor, hindering its widespread adoption. Moreover, conventional lithographic methods primarily yield 2D planar devices, thereby limiting the selection of materials and geometrical configurations. To address these challenges, this work introduces a streamlined fabrication method for 3D spiral microfluidic devices, employing rotational force within a miniaturized thermal drawing process, termed as mini-rTDP. This innovation allows for rapid prototyping of twisted fiber-based microfluidics featuring versatility in material selection and heightened geometric intricacy. To validate the performance of these devices, we combined computational modeling with microtomographic particle image velocimetry (µTPIV) to comprehensively characterize the 3D flow dynamics. Our results corroborate the presence of a steady secondary flow, underscoring the effectiveness of our approach. Our 3D spiral microfluidics platform paves the way for exploring intricate microflow dynamics, with promising applications in areas such as drug delivery, diagnostics, and lab-on-a-chip systems.

Passive microfluidic devices for cell separation.

The separation of specific cell populations is instrumental in gaining insights into cellular processes, elucidating disease mechanisms, and advancing applications in tissue engineering, regenerative medicine, diagnostics, and cell therapies. Microfluidic methods for cell separation have propelled the field forward, benefitting from miniaturization, advanced fabrication technologies, a profound understanding of fluid dynamics governing particle separation mechanisms, and a surge in interdisciplinary investigations focused on diverse applications. Cell separation methodologies can be categorized according to their underlying separation mechanisms. Passive microfluidic separation systems rely on channel structures and fluidic rheology, obviating the necessity for external force fields to facilitate label-free cell separation. These passive approaches offer a compelling combination of cost-effectiveness and scalability when compared to active methods that depend on external fields to manipulate cells. This review delves into the extensive utilization of passive microfluidic techniques for cell separation, encompassing various strategies such as filtration, sedimentation, adhesion-based techniques, pinched flow fractionation (PFF), deterministic lateral displacement (DLD), inertial microfluidics, hydrophoresis, viscoelastic microfluidics, and hybrid microfluidics. Besides, the review provides an in-depth discussion concerning cell types, separation markers, and the commercialization of these technologies. Subsequently, it outlines the current challenges faced in the field and presents a forward-looking perspective on potential future developments. This work hopes to aid in facilitating the dissemination of knowledge in cell separation, guiding future research, and informing practical applications across diverse scientific disciplines.

Recombinant adeno-associated virus production evaluation in Chinese hamster ovary cells.

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.

Utilizing targeted integration CHO pools to potentially accelerate the GMP manufacturing of monoclonal and bispecific antibodies.

Monoclonal antibodies (mAbs) are effective therapeutic agents against many acute infectious diseases including COVID-19, Ebola, RSV, Clostridium difficile, and Anthrax. mAbs can therefore help combat a future pandemic. Unfortunately, mAb development typically takes years, limiting its potential to save lives during a pandemic. Therefore "pandemic mAb" timelines need to be shortened. One acceleration tool is "deferred cloning" and leverages new Chinese hamster ovary (CHO) technology based on targeted gene integration (TI). CHO pools, instead of CHO clones, can be used for Phase I/II clinical material production. A final CHO clone (producing the mAb with a similar product quality profile and preferably with a higher titer) can then be used for Phase III trials and commercial manufacturing. This substitution reduces timelines by ~3 months. We evaluated our novel CHO TI platform to enable deferred cloning. We created four unique CHO pools expressing three unique mAbs (mAb1, mAb2, and mAb3), and a bispecific mAb (BsAb1). We then performed single-cell cloning for mAb1 and mAb2, identifying three high-expressing clones from each pool. CHO pools and clones were inoculated side-by-side in ambr15 bioreactors. CHO pools yielded mAb titers as high as 10.4 g/L (mAb3) and 7.1 g/L (BsAb1). Subcloning yielded CHO clones expressing higher titers relative to the CHO pools while yielding similar product quality profiles. Finally, we showed that CHO TI pools were stable by performing a 3-month cell aging study. In summary, our CHO TI platform can increase the speed to clinic for a future "pandemic mAb."

Rheological effects on purely-elastic flow asymmetries in the cross-slot geometry.

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.

Alignment-Rheology Relationship of Biosourced Rod-Like Colloids and Polymers under Flow.

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.