Cloaked Exosomes: Biocompatible, Durable, and Degradable Encapsulation.

Exosomes-nanosized extracellular vesicles (EVs) naturally secreted from cells-have emerged as promising biomarkers and potential therapeutic vehicles, but methods to manipulate them for engineering purposes remain elusive. Among the technical obstacles are the small size and surface complexity of exosomes and the complex processing steps required, which reduce the biocompatibility of currently available methods. The encapsulation of exosomes with a nanofilm of supramolecular complexes of ferric ions (Fe3+ ) and tannic acid is demonstrated here. The resulting natural polyphenol, ≈10 nm thick, protects exosomes from external aggressors such as UV-C irradiation or heat and is controllably degraded on demand. Furthermore, gold nanoparticles can be covalently attached for single-exosome level visualization. To fully exploit their therapeutic potential, chemotherapeutic drug-loaded EVs are functionalized to achieve the targeted, selective killing of cancer cells preferentially over normal cells. This nanofilm not only preserves the native size and chemical makeup of the intrinsic exosomes, but also confers new capabilities for efficient tumor targeting and pH-controlled release of drugs. Demonstrating a scalable method to produce biocompatible, durable, on-demand degradable, and chemically controllable shields for exosome modification and functionalization, the methods introduced here are expected to bring the potential of exosome-based nanomedicine applications closer to reality.

Platelet Membrane-Enclosed Bioorthogonal Catalysis for Combating Dental Caries.

Platelets have shown promise as a means to combat bacterial infections, fostering the development of innovative therapeutic approaches. However, several challenges persist, including cargo loading issues, limited efficacy against biofilms, and concerns regarding the impact of payloads on the platelet carriers. Here, human platelet membrane vesicles (h-PMVs) encapsulating supramolecular metal catalysts (SMCs) as "nanofactories" to convert prodrugs into antimicrobial compounds within close proximity to bacteria are introduced. Having established the feasibility and effectiveness of the SMCs within h-PMVs, referred to as the PLT-reactor, to activate pro-antibiotic drugs (pro-ciprofloxacin and pro-moxifloxacin) using model organisms (Escherichia coli ATCC 25922 and Staphylococcus aureus ATCC 25923), the investigation is subsequently extended to oral biofilms, with a particular emphasis on Streptococcus mutans 3065. This "bind and kill" strategy demonstrates the potent antimicrobial specificity of the PLT-reactor through localized antibiotic production. h-PMVs play a pivotal role by enabling precise targeting of pathogenic biofilms on natural teeth while minimizing potential hemolytic effects. The finding indicates that platelet membrane-cloaked surfaces exhibit robust, multifaceted, and pathogen-specific binding affinity with excellent biocompatibility, making them a promising alternative to antibody-based therapies for infectious diseases.

Productive chemical interaction between a bacterial microcolony couple is enhanced by periodic relocation.

This paper describes a system to study how small physical perturbations can affect bacterial community behavior in unexpected ways through modulation of diffusion and convective transport of chemical communication molecules and resources. A culture environment that mimics the chemically open characteristic of natural bacterial habitats but with user-defined spatiotemporal control of bacteria microcolonies is realized through use of an aqueous two phase system (ATPS). The ATPS is formulated with nontoxic dextran (DEX) and poly(ethylene glycol) (PEG) dissolved in cell culture media. DEX-phase droplets formed within a bulk PEG-phase stably confine the bacteria within it while small molecules diffuse relatively freely. Bacteria-containing DEX droplets can also be magnetically relocated, without loss of its bacterial content, when DEX-conjugated magnetic particles are included. We found that decreasing the distance between quorum-sensing (QS)-coupled microcolonies increased green fluorescent protein (GFP) expression due to increased inter-colony chemical communication but with upper limits. Periodic relocation of the chemical signal receiver colony, however, increased GFP expression beyond these typical bounds predicted by quorum sensing concepts alone by maintaining inter-colony chemical communication while also relieving the colony of short-range resource depletion effects. Computer simulations suggest that such increased productive output in response to periodic nonlethal physical perturbations is a common feature of chemically activated interactive cell systems where there is also a short-range inhibitory effect. In addition to providing insights on the effect of bacteria relocation, the magnetic ATPS droplet manipulation capability should be broadly useful for bioanalyses applications where selective partitioning at the microscale in fully aqueous conditions is needed.

Lab-on-a-disc for fully integrated multiplex immunoassays.

This paper presents a cost-effective, rapid, and fully automated lab-on-a-disc for simultaneous detection of multiple protein biomarkers in raw samples such as whole blood or whole saliva. For the diagnosis of cardiovascular disease, here, a novel centrifugal microfluidic layout was designed to conduct the simultaneous detection of high sensitivity C-reactive protein, cardiac troponin I, and N-terminal pro-B type natriuretic peptide based on a bead-based sandwich type enzyme-linked immunosorbent assay (ELISA). Three reaction chambers are initially interconnected for the common processes such as sample injection, incubation, and washing and then isolated on-demand for the independent processes such as substrate incubation and final detection. The assay performances such as the limit of detection and the dynamic range were comparable with those of the conventional ELISA despite the significant reduction of the minimum sample volume (200 µL), the amount of washing buffer (700 µL), and the total process time (20 min).

Aqueous two-phase system-derived biofilms for bacterial interaction studies.

We describe patterning of bacterial biofilms using polymer-based aqueous two-phase system (ATPS) microprinting protocols. The fully aqueous but selectively bacteria-partitioning nature of the ATPS allows spatially distinct localization of suspensions of bacteria such as Pseudomonas aeruginosa and Escherichia coli with high precision. The ATPS patterned bacterial suspensions form spatially distinct biofilms over time. Due to the fully aqueous and gentle noncontact printing procedures employed, coculture biofilms composed of multiple types of bacteria could be printed not only adjacent to each other but also directly over another layer of existing biofilm. In addition, the ATPS environment also allows free diffusion of small molecules between spatially distinct and localized bacterial suspensions and biofilms. This enables biofilms to chemically affect or be affected by neighboring biofilms or planktonic cells, even if they consist of different strains or species. We show that a ß-lactamase producing biofilm confers ampicillin resistance to neighboring nonresistant planktonic cells, as seen by a 3,600-fold increase in survival of the ampicillin-sensitive strain. These examples demonstrate the ability of ATPS-based biofilm patterning methods to enable unique studies on commensalistic effects between bacterial species.

Flexible fabrication and applications of polymer nanochannels and nanoslits.

Fluidic devices that employ nanoscale structures (<100 nm in one or two dimensions, slits or channels, respectively) are generating great interest due to the unique properties afforded by this size domain compared to their micro-scale counterparts. Examples of interesting nanoscale phenomena include the ability to preconcentrate ionic species at extremely high levels due to ion selective migration, unique molecular separation modalities, confined environments to allow biopolymer stretching and elongation and solid-phase bioreactions that are not constrained by mass transport artifacts. Indeed, many examples in the literature have demonstrated these unique opportunities, although predominately using glass, fused silica or silicon as the substrate material. Polymer microfluidics has established itself as an alternative to glass, fused silica, or silicon-based fluidic devices. The primary advantages arising from the use of polymers are the diverse fabrication protocols that can be used to produce the desired structures, the extensive array of physiochemical properties associated with different polymeric materials, and the simple and robust modification strategies that can be employed to alter the substrate's surface chemistry. However, while the strengths of polymer microfluidics is currently being realized, the evolution of polymer-based nanofluidics has only recently been reported. In this critical review, the opportunities afforded by polymer-based nanofluidics will be discussed using both elastomeric and thermoplastic materials. In particular, various fabrication modalities will be discussed along with the nanometre size domains that they can achieve for both elastomer and thermoplastic materials. Different polymer substrates that can be used for nanofluidics will be presented along with comparisons to inorganic nanodevices and the consequences of material differences on the fabrication and operation of nanofluidic devices (257 references).

Simple replication methods for producing nanoslits in thermoplastics and the transport dynamics of double-stranded DNA through these slits.

Mixed-scale nano- and microfluidic networks were fabricated in thermoplastics using simple and robust methods that did not require the use of sophisticated equipment to produce the nanostructures. High-precision micromilling (HPMM) and photolithography were used to generate mixed-scale molding tools that were subsequently used for producing fluidic networks into thermoplastics such as poly(methyl methacrylate), PMMA, cyclic olefin copolymer, COC, and polycarbonate, PC. Nanoslit arrays were imprinted into the polymer using a nanoimprinting tool, which was composed of an optical mask with patterns that were 2-7 µm in width and a depth defined by the Cr layer (100 nm), which was deposited onto glass. The device also contained a microchannel network that was hot embossed into the polymer substrate using a metal molding tool prepared via HPMM. The mixed-scale device could also be used as a master to produce a polymer stamp, which was made from polydimethylsiloxane, PDMS, and used to generate the mixed-scale fluidic network in a single step. Thermal fusion bonding of the cover plate to the substrate at a temperature below their respective T(g) was accomplished by oxygen plasma treatment of both the substrate and cover plate, which significantly reduced thermally induced structural deformation during assembly: ∼6% for PMMA and ∼9% for COC nanoslits. The electrokinetic transport properties of double-stranded DNA (dsDNA) through the polymeric nanoslits (PMMA and COC) were carried out. In these polymer devices, the dsDNA demonstrated a field-dependent electrophoretic mobility with intermittent transport dynamics. DNA mobilities were found to be 8.2 ± 0.7 × 10(-4) cm(2) V(-1) s(-1) and 7.6 ± 0.6 × 10(-4) cm(2) V(-1) s(-1) for PMMA and COC, respectively, at a field strength of 25 V cm(-1). The extension factors for λ-DNA were 0.46 in PMMA and 0.53 in COC for the nanoslits (2-6% standard deviation).

Highly sensitive detection of hydrazine by a disposable, Poly(Tannic Acid)-Coated carbon electrode.

We propose an electrochemical sensor based on the enhanced electrocatalytic oxidation exhibited on a functionalized poly(tannic acid) coating to detect hydrazine. Tannic acid, a naturally abundant and low-cost polyphenol, was enzymatically polymerized with horseradish peroxidase and subsequently adsorbed on a disposable screen-printed carbon electrode with a short incubation time (30 min). The fabrication method proved to be reproducible (4.2 % relative standard deviation), with the sensors displaying high sensitivity (7 × 10-3 µA mm-2 µM-1) and selectivity even in the presence of various common interfering agents. The low detection limit (100 nM) and robustness of the sensor demonstrated its suitability for environmental applications. It can be used to quantify hydrazine in tap and river water samples.

Bacteria concentration using a membrane type insulator-based dielectrophoresis in a plastic chip.

We report an insulator-based (or, electrodeless) dielectrophoresis utilizing microfabricated plastic membranes. The membranes with honeycomb-type pores have been fabricated by patterning the SU-8 layer on a substrate which was pretreated with self-assembled monolayer of octadecyltrichlorosilane for the easy release. The fabricated membrane was positioned between two electrodes and alternating current field was applied for the particle trap experiments. The particle could be trapped due to the dielectrophoresis force generated by the non-uniformities of the electric fields applied through the membranes with pores. Simulations using CFD-ACE+(CFD Research, Huntsville, Alabama) suggested that the dielectrophoresis force is stronger in the edge of the pores where the field gradient is highest. The bacteria could be captured on the near edge of the pores when the electric field was turned on and the trapped bacteria could be released when the field was turned off with the release efficiency of more than 93+/-7%. The maximal trapping efficiency of 66+/-7% was obtained under the electric fields (E=128 V/mm and f=300 kHz) when the dilute bacteria solution (Escherichia coli: 9.3 x 10(3) cell/mL, 0.5 mS/m) flowed with a flow rate of 100 microL/min.

Biologically-active unilamellar vesicles from red blood cells.

We demonstrate a method to prepare giant unilamellar vesicles (GUVs) with biologically-active protein activity, by mixing erythrocyte (red blood cell) membrane extract with phospholipids and growing their mixture in a porous hydrogel matrix. This presents a pathway to retain protein biological activity without prior isolation and purification of the protein, though only the activity of the membrane protein GLUT1 is investigated to date. Using the cascade enzymatic reaction glucose oxidase and horseradish peroxidase to assay glucose concentration specifically within the GUV interior, we show that glucose is internalized by GLUT1 whereas adding cytochalasin B, a GLUT1 inhibitor, blocks glucose transport. The method presented here operates at biological ionic strength and is both simple and potentially generalizable.

Surface-Engineered Paper Hanging Drop Chip for 3D Spheroid Culture and Analysis.

Protein corona coated onto the hydrophilic cellulose fiber turns into hydrophobic upon UV irradiation without hindering the porosity of the paper while simultaneously reducing nonspecific adsorption. Taking advantage of the biofouling-resistant, hydrophobic, and fluid transport through property, we demonstrated hanging drop three-dimensional (3D) spheroid culture and in-site analysis, including drug testing, time-dependent detection of secreted protein, and fluorescence staining without disturbing the spheroids. This single hanging drop system can also be extended to a networked hanging drop chip to mimic in vivo microphysiology by combining with wax-patterned microfluidic channels, where well-to-well interaction can be accurately controlled in a passive manner. As a proof of concept, the effects of a concentration gradient of nutrient and variable dosage of anticancer drugs were studied in the 3D spheroids cultured on paper. The experimental results suggested that a complex network device could be fabricated on a large scale on demand at a minimal cost for 3D spheroid culture. Our method demonstrates a future possibility for paper as a low cost, high-throughput 3D spheroid-based "body-on-a-chip" platform material.

A lab-on-a-disc with reversible and thermally stable diaphragm valves.

A lab-on-a-disc is a unique microfluidic platform that utilizes centrifugal force to pump liquids. This offers many benefits for point-of-care devices because it eliminates the need for connections to multiple pumps and complex tubing connections. A wide range of applications including clinical chemistry, immunoassay, cell analysis, and nucleic acid tests could be demonstrated on a spinning disc. To enable the performance of assays in a fully integrated and automated manner, the robust actuation of integrated valves is a prerequisite. However, conventional passive-type valves incur a critical drawback in that their operation is dependent on the rotational frequency, which is easily influenced by the channel geometry and chemistry, in addition to the physical properties of the liquids to be transferred. Even though a few active-type valving techniques permit the individual actuation of valves, independent of the rotational frequency, complex procedures for the fabrication as well as actuation mechanisms have prevented their broader acceptance in general applications. Here, we report on a lab-on-a-disc incorporating individually addressable diaphragm valves (ID valves) that enable the reversible and thermally stable actuation of multiple valves with unprecedented ease and robustness. These ID valves are configured from an elastic epoxy diaphragm embedded on a 3D printed push-and-twist valve, which can be easily actuated by a simple automatic driver unit. As a proof of concept experiment, an enzyme-linked immunosorbent assay (ELISA) and a polymerase chain reaction (PCR) were performed on a disc in a fully automated manner to demonstrate the robust, reversible, leak-free, and thermally stable actuation of the valves.

Aminosilane layers on the plasma activated thermoplastics: influence of solvent on its structure and morphology.

The chemistry and the structure of aminosilane layer on the plasma activated thermoplastic substrates, e.g., polycarbonate (PC), polystyrene (PS), poly(methyl methacrylate) (PMMA), and cyclic olefin co-polymer (COC) were investigated at the molecular level. The nature of the surface functional groups of the silane layers prepared by solution phase deposition in aqueous and anhydrous solvents were studied using various techniques including ellipsometry, goniometry, atomic force microscopy (AFM), X-ray photoelectron spectroscopy (XPS), and attenuated total reflectance infrared spectroscopy (ATR-IR). The XPS analyses revealed the presence of various oxygen functionalities on the plasma activated thermoplastics. Considerable differences were observed for the structure of aminosilane depending on the solvent used for the reaction. Deposition from aqueous solution resulted in relatively flat and smooth surfaces with consistent thickness compared to the anhydrous solution deposition. In the former case, 33% of the total nitrogen accounted for protonated amine and 16% for the free amino groups. In the latter, only 6% accounted for the protonated amine. The point of zero charge (pzc), on the aminosilane modified PC was found to be around 7, indicated that the surface is positively charged below pH 7 and negatively charged above pH 7. The surface analysis data suggested that various interactions are possible between the plasma activated thermoplastic surface and the aminosilane. In general, they are bound to the surface through covalent bond formation between the oxygen functionalities on the thermoplastic surface and the amino or the silanol groups of the aminosilane.

Elastomeric membrane valves in a disc.

We present elastomeric membrane valves integrated into a centrifugal microfluidic platform for precise control of fluid on a disc. The amount of the fluid passing through the valves, which depends on the rotating speed of the disc and the membrane thickness, has been characterized, and could be precisely controlled by tuning the disc motion.

Simple room temperature bonding of thermoplastics and poly(dimethylsiloxane).

We describe a simple and versatile method for bonding thermoplastics to elastomeric polydimethylsiloxane (PDMS) at room temperature. The bonding of various thermoplastics including polycarbonate (PC), cyclic olefin copolymer (COC), polymethylmethacrylate (PMMA), and polystyrene (PS), to PDMS has been demonstrated at room temperature. An irreversible bonding was formed instantaneously when the thermoplastics, activated by oxygen plasma followed by aminopropyltriethoxysilane modification, were brought into contact with the plasma treated PDMS. The surface modified thermoplastics were characterized by water contact angle measurements and X-ray photoelectron spectroscopy. The tensile strength of the bonded hybrid devices fabricated with PC, COC, PMMA, and PS was found to be 430, 432, 385, and 388 kPa, respectively. The assembled devices showed high burst resistance at a maximum channel pressure achievable by an in-house built syringe pump, 528 kPa. Furthermore, they displayed very high hydrolytic stability; no significant change was observed even after the storage in water at 37 °C over a period of three weeks. In addition, this thermoplastic-to-PDMS bonding technique has been successfully employed to fabricate a relatively large sized device. For example, a lab-on-a-disc with a diameter of 12 cm showed no leakage when it spins for centrifugal fluidic pumping at a very high rotating speed of 6000 rpm.