Multifunctional covalently stabilized vesicles acting simultaneously as the template of gold nanoparticle cluster and the nanocarrier of guest molecules

The terminal hydroxyl groups of amphiphilic multiarm star copolymers with a hydrophilic hyperbranched polyethylenimine (PEI) core and hydrophobic poly(ε-caprolactone) (PCL) arms were partially or completely transformed into the radical-crosslinkable methacrylate (MA) groups (PEI-b-PCL-MA). The resulting PEI-b-PCL-MA polymers with 100% MA substitution self-assembled in water into simple vesicles, whereas those with partial MA substitution aggregated into complex vesicles. These structures could be proved by transmission electron microscopy and dynamic light scattering only after crosslinking the intra-vesicular MA groups that generated the covalently stabilized vesicles (CSVs). The obtained CSVs could be used as host for the formation of gold nanoparticle (AuNP) cluster, and the AuNP clusters stabilized by the CSVs were stable under a wider range of CSV/AuNP feed ratio than those stabilized by the uncrosslinked precursors. The diameter of AuNPs in the clusters was in the range of 4-6 nm, and the distance of adjacent AuNPs could be modulated through altering the feed ratio of CSV/AuNP. The color of the solutions of AuNPs with CSV could be tuned from brown to red, purple, even blue. The composites of CSV and AuNPs could be further used as nanocarriers to accommodate hydrophobic guest of pyrene, and a higher amount of AuNPs in the nanocarriers led to a lower encapsulation capacity for pyrene guests.     

Cellular microarrays for use with capillary-driven microfluidics

We present a method for the facile arraying of cells on microstructured substrates which should be suitable for cellular assays in autonomous microfluidic capillary systems (CSs). The CSs, which were designed and microfabricated in Si, have various microfluidic functional elements including reaction chambers wherein cellular arrays are located. Two methods for arraying the cells were explored. In the first method, a hydrophobic alkanethiol was microcontact-printed on the bottom surface of a microfluidic reaction chamber. The subsequent adsorption of protein-repellent alkanethiols around the printed areas and the deposition from solution of fibronectin (FN) on the hydrophobic areas resulted in an adhesive pattern for the attachment of living human breast cancer cells. This method was limited by the formation of cellular clusters, which proved difficult to remove selectively. The second method employed a poly(dimethylsiloxane) elastomer having oval recessed microstructures. The selective coating of the inner walls of the ovals with FN and the blocking of the mesas around the ovals with bovine serum albumin (BSA) permitted single or multiple cells to be arrayed depending on the size of the ovals. The possibility of sealing CSs with cells arrayed on poly(dimethylsiloxane) may provide a versatile platform for high-throughput experimentation down to the single-cell level. Figure The deposition of one or a few living cells in fibronectin-coated poly(dimethylsiloxane) microstructures results in cellular arrays, which can be interfaced with capillary-driven microfluidics     

Continuous flow in open microfluidics using controlled evaporation

This paper presents a method for programming the flow rate of liquids inside open microfluidic networks (MFNs). A MFN comprises a number of independent flow paths, each of which starts with an open filling port, has a sealed microchannel in which assays can be performed, and an open capillary pump (CP). The MFN is placed over Peltier elements and its flow paths initially fill owing to capillary forces when liquids are added to the filling ports. A cooling Peltier element underneath the filling ports dynamically prevents evaporation in all filling ports using the ambient temperature and relative humidity as inputs. Another Peltier element underneath the CPs heats the pumps thereby inducing evaporation in the CPs and setting the flow rate in the microchannels. This method achieves flow rates in the microchannels ranging from approximately 1.2 nL s(-1) to approximately 30 pL s(-1), and is able to keep 90% of a 0.6 microL solution placed in an open filling port for 60 min. This simple and efficient method should be applicable to numerous assays or chemical reactions that require small and precise flow of liquids and reagents inside microfluidics.     

Microcontact printing of proteins inside microstructures

Microfluidic devices are well suited for the miniaturization of biological assays, in particular when only small volumes of samples and reagents are available, short time to results is desirable, and multiple analytes are to be detected. Microfluidic networks (MFNs), which fill by means of capillary forces, have already been used to detect important biological analytes with high sensitivity and in a combinatorial fashion. These MFNs were coated with Au, onto which a hydrophilic, protein-repellent monolayer of thiolated poly(ethyleneglycol) (HS-PEG) was self-assembled, and the binding sites for analytes were present on a poly(dimethylsiloxane) (PDMS) sealing cover. We report here a set of simple methods to extend previous work on MFNs by integrating binding sites for analytes inside the microstructures of MFNs using microcontact printing (muCP). First, fluorescently labeled antibodies (Abs) were microcontact-printed from stamps onto planar model surfaces such as glass, Si, Si/SiO2, Au, and Au derivatized with HS-PEG to investigate how much candidate materials for MFNs would quench the fluorescence of printed, labeled Abs. Au coated with HS-PEG led to a fluorescence signal that was approximately 65% weaker than that of glass but provided a convenient surface for printing Abs and for rendering the microstructures of the MFNs wettable. Then, proteins were inked from solution onto the surface of PDMS (Sylgard 184) stamps having continuous or discontinuous micropatterns or locally inked onto planar stamps to investigate how the aspect ratio (depth:width) of microstructures and the printing conditions affected the transfer of protein and the accuracy of the resulting patterns. By applying a controlled pressure to the back of the stamp, Abs were accurately microcontact-printed into the recessed regions of MFNs if the aspect ratio of the MFN microstructures was lower than approximately 1:6. Finally, the realization of a simple assay between Abs (used as antigens) microcontact-printed in microchannels and Abs from solution suggests that this method could become useful to pattern proteins in microstructures for advanced bioanalytical purposes.     

Capillary pumps for autonomous capillary systems

Autonomous capillary systems (CSs), where liquids are displaced by means of capillarity, are efficient, fast and convenient platforms for many bioanalytical applications. The proper functioning of these microfluidic devices requires displacing accurate volumes of liquids with precise flow rates. In this work, we show how to design capillary pumps for controlling the flow properties of CSs. The capillary pumps comprise microstructures of various shapes with dimensions from 15-250 microm, which are positioned in the capillary pumps to encode a desired capillary pressure. The capillary pumps are designed to have a small flow resistance and are preceded by a constricted microchannel, which acts as a flow resistance. Therefore, both the capillary pump and the flow resistance define the flow rate in the CS, and flow rates from 0.2-3.7 nL s(-1) were achieved. The placement and the shape of the microstructures in the capillary pumps are used to tailor the filling front of liquids in the capillary pumps to obtain a reliable filling behaviour and to minimize the risk of entrapping air. The filling front can, for example, be oriented vertically or tilted to the main axis of the capillary pump. We also show how capillary pumps having different hydrodynamic properties can be connected to program a sequence of slow and fast flow rates in a CS.     

Single‐step CE for miniaturized and easy‐to‐use system

We developed a novel single‐step capillary electrophoresis (SSCE) scheme for miniaturized and easy to use system by using a microchannel chip, which was made from the hydrophilic material polymethyl methacrylate (PMMA), equipped with a capillary stop valve. Taking the surface tension property of liquids into consideration, the capillary effect was used to introduce liquids and control capillary stop valves in a partial barrier structure in the wall of the microchannel. Through the combined action of stop valves and air vents, both sample plug formation for electrophoresis and sample injection into a separation channel were successfully performed in a single step. To optimize SSCE, different stop valve structures were evaluated using actual microchannel chips and the finite element method with the level set method. A partial barrier structure at the bottom of the channel functioned efficiently as a stop valve. The stability of stop valve was confirmed by a shock test, which was performed by dropping the microchannel chip to a floor. Sample plug deformation could be reduced by minimizing the size of the side partial barrier. By dissolving hydroxyl ethyl cellulose and using it as the sample solution, the EOF and adsorption of the sample into the PMMA microchannel were successfully reduced. Using this method, a 100‐bp DNA ladder was concentrated; good separation was observed within 1 min. At a separation length of 5 mm, the signal was approximately 20‐fold higher than a signal of original sample solution by field‐amplified sample stacking effect. All operations, including liquid introduction and sample separation, can be completed within 2 min by using the SSCE scheme.     

Capillary-driven flow in corner geometries

Wetting of corner-containing geometries is ubiquitous, since the man-made surfaces and natural surfaces are usually not atomically smooth and contain pores, grooves, and cracks. In spite of the very long history of the research of capillary phenomena, the most attention was paid to capillary rise in cylindrical capillaries leaving the rich physics of the capillary transport of the liquids in the corner geometries unravelled. The present work aims to review the progress in studying of wetting of corner-containing geometries: isolated corners, rectangular channels, and confined angular geometries. The review is believed to be of interest for readers from fields such as oil and gas industry, space science, biophysics, and microfluidics.     

Microfluidic platforms for lab-on-a-chip applications

We review microfluidic platforms that enable the miniaturization, integration and automation of biochemical assays. Nowadays nearly an unmanageable variety of alternative approaches exists that can do this in principle. Here we focus on those kinds of platforms only that allow performance of a set of microfluidic functions--defined as microfluidic unit operations-which can be easily combined within a well defined and consistent fabrication technology to implement application specific biochemical assays in an easy, flexible and ideally monolithically way. The microfluidic platforms discussed in the following are capillary test strips, also known as lateral flow assays, the "microfluidic large scale integration" approach, centrifugal microfluidics, the electrokinetic platform, pressure driven droplet based microfluidics, electrowetting based microfluidics, SAW driven microfluidics and, last but not least, "free scalable non-contact dispensing". The microfluidic unit operations discussed within those platforms are fluid transport, metering, mixing, switching, incubation, separation, droplet formation, droplet splitting, nL and pL dispensing, and detection.     

Stable and Oriented Liquid Crystal Droplets Stabilized by Imidazolium Ionic Liquids

Liquid crystals represent a fascinating intermediate state of matter, with dynamic yet organized molecular features and untapped opportunities in sensing. Several works report the use of liquid crystal droplets formed by microfluidics and stabilized by surfactants such as sodium dodecyl sulfate (SDS). In this work, we explore, for the first time, the potential of surface-active ionic liquids of the imidazolium family as surfactants to generate in high yield, stable and oriented liquid crystal droplets. Our results show that [C₁₂MIM][Cl], in particular, yields stable, uniform and monodisperse droplets (diameter 74 ± 6 µm; PDI = 8%) with the liquid crystal in a radial configuration, even when compared with the standard SDS surfactant. These findings reveal an additional application for ionic liquids in the field of soft matter.     

Design and fabrication of chemically robust three-dimensional microfluidic valves

A current problem in microfluidics is that poly(dimethylsiloxane) (PDMS), used to fabricate many microfluidic devices, is not compatible with most organic solvents. Fluorinated compounds are more chemically robust than PDMS but, historically, it has been nearly impossible to construct valves out of them by multilayer soft lithography (MSL) due to the difficulty of bonding layers made of "non-stick" fluoropolymers necessary to create traditional microfluidic valves. With our new three-dimensional (3D) valve design we can fabricate microfluidic devices from fluorinated compounds in a single monolithic layer that is resistant to most organic solvents with minimal swelling. This paper describes the design and development of 3D microfluidic valves by molding of a perfluoropolyether, termed Sifel, onto printed wax molds. The fabrication of Sifel-based microfluidic devices using this technique has great potential in chemical synthesis and analysis.     

Fabrication of high-aspect-ratio polymer microstructures and hierarchical textures using carbon nanotube composite master molds

Scalable and cost effective patterning of polymer structures and their surface textures is essential to engineer material properties such as liquid wetting and dry adhesion, and to design artificial biological interfaces. Further, fabrication of high-aspect-ratio microstructures often requires controlled deep-etching methods or high-intensity exposure. We demonstrate that carbon nanotube (CNT) composites can be used as master molds for fabrication of high-aspect-ratio polymer microstructures having anisotropic nanoscale textures. The master molds are made by growth of vertically aligned CNT patterns, capillary densification of the CNTs using organic solvents, and capillary-driven infiltration of the CNT structures with SU-8. The composite master structures are then replicated in SU-8 using standard PDMS transfer molding methods. By this process, we fabricated a library of replicas including vertical micro-pillars, honeycomb lattices with sub-micron wall thickness and aspect ratios exceeding 50:1, and microwells with sloped sidewalls. This process enables batch manufacturing of polymer features that capture complex nanoscale shapes and textures, while requiring only optical lithography and conventional thermal processing.     

Structure and function of proteins in hydrated choline dihydrogen phosphate ionic liquid

Ionic liquids are being intensely studied as promising media for the stabilization of proteins and other biomolecules. Choline dihydrogen phosphate (CDHP) has been identified as one of the most promising candidates for this application. In this work we have probed in more detail the effects that CDHP may have on the thermodynamics, structure, and stability of proteins, including one of therapeutic interest. Microcalorimetry and circular dichroism spectropolarimetry (CD) were used to assess the thermal stability of protein solutions in CDHP/water mixtures at various concentrations. Increasing thermal stability of lysozyme and interleukin-2 in proportion to CDHP concentration was observed. Isothermal titration calorimetry (ITC) was used to quantify binding interactions, and indicate that the mechanism for stability does not appear to be dependent upon CDHP binding to protein. CD and small angle X-ray scattering (SAXS) analyses were used to probe for structural changes due to the presence of CDHP. SAXS indicates charge effects on the surface of the protein play a role in protein stability in ionic liquids, and no significant alteration of the overall tertiary conformation of lysozyme was observed at 25 °C. However, after incubation at 37 °C or at higher concentrations of CDHP, small changes in protein structure were seen. Effects on protein activity were monitored using turbidity assays, and CDHP decreases protein activity but does not eliminate it. Protein solubility was also monitored using a turbidity assay and was found to be inversely proportional to the concentration of CDHP in solution.     

The role of growth temperature in the adhesion and mechanics of pathogenic L. monocytogenes: an AFM study

The adhesion strengths of pathogenic L. monocytogenes EGDe to a model surface of silicon nitride were quantified using atomic force microscopy (AFM) in water for cells grown under five different temperatures (10, 20, 30, 37, and 40 °C). The temperature range investigated was chosen to bracket the thermal conditions in which L. monocytogenes survive in the environment. Our results indicated that adhesion force and energy quantified were at their maximum when the bacteria were grown at 30 °C. The higher adhesion observed at 30 °C compared to the adhesion quantified for bacterial cells grown at 37, 40, 20, and 10 °C was associated with longer and denser bacterial surface biopolymer brushes as predicted from fitting a model of steric repulsion to the approach distance-force data as well from the results of protein colorimetric assays. Theoretically predicted adhesion energies based on soft-particle DLVO theory agreed well with the adhesion energies computed from AFM force-distance retraction data (r(2) = 0.94); showing a minimum energy barrier to adhesion at 30 °C.     

Heterogeneous critical nucleation on a completely wettable substrate

Heterogeneous nucleation of a new bulk phase on a flat substrate can be associated with the surface phase transition called wetting transition. When this bulk heterogeneous nucleation occurs on a completely wettable flat substrate with a zero contact angle, the classical nucleation theory predicts that the free-energy barrier of nucleation vanishes. In fact, there always exists a critical nucleus and a free-energy barrier as the first-order prewetting transition will occur even when the contact angle is zero. Furthermore, the critical nucleus changes its character from the critical nucleus of surface phase transition below bulk coexistence (undersaturation) to the critical nucleus of bulk heterogeneous nucleation above the coexistence (oversaturation) when it crosses the coexistence. Recently, Sear [J. Chem. Phys. 129, 164510 (2008)] has shown, by a direct numerical calculation of nucleation rate, that the nucleus does not notice this change when it crosses the coexistence. In our work, the morphology and the work of formation of critical nucleus on a completely wettable substrate are re-examined across the coexistence using the interface-displacement model. Indeed, the morphology and the work of formation changes continuously at the coexistence. Our results support the prediction of Sear and will rekindle the interest on heterogeneous nucleation on a completely wettable substrate.     

Pump Effect of a Capillary Discharge in Electrically Conductive Liquids

Among the configurations to generate plasma in electrically conductive liquids only the diaphragm and the capillary discharge schemes allow to generate plasma which is not in contact with one of the electrodes. Based on this concept, this work reports for the first time the development of an underwater plasma pump, in which the periodic electrical breakdown inside an asymmetrical (sub-)millimetre hole results in a net flow of aqueous solution through the hole without the use of any moving parts such as valves or diaphragms typically used in micropumps. Certain capillary geometries feature very stable flow rates and even allow altering flow direction by changing the power. By varying the hole’s dimensions, the range of time-independent flow rates covers more than one order of magnitude and as the discharge produces some of the strongest oxidants available, we believe that this concept might find application in fields as water decontamination and sterilization.     

Capillary penetration failure of blood suspensions

Blood suspension fails to penetrate a capillary with radius R less than 50 microm even if the capillary is perfectly wettable. This invasion threshold is attributed to three red blood cells (RBCs) segregation mechanisms--corner deflection at the entrance, the intermediate deformation-induced radial migration and shear-induced diffusion within a packed slug at the meniscus. The shear-induced radial migration for deformable particles endows the blood cells with a higher velocity than the meniscus to form the concentrated slug behind the meniscus. This tightly packed slug has a higher resistance and arrests the flow. Rigid particles and rigidified blood cells result in wetting behavior similar to that seen for homogeneous liquids, with decreased RBC migration towards the capillary centerline and reduction of packing. Corner deflection with a radial drift velocity accelerates the radial migration for small capillaries. However, deformation-induced radial migration is the key mechanism responsible for penetration failure. This sequence of mechanisms is confirmed through videomicroscopy and scaling theories were applied to capture the dependence of the critical capillary radius as a function of RBC concentrations.     

Controlled release of reagents in capillary-driven microfluidics using reagent integrators

The integration and release of reagents in microfluidics as used for point-of-care testing is essential for an easy and accurate operation of these promising diagnostic devices. Here, we present microfluidic functional structures, which we call reagent integrators (RIs), for integrating and releasing small amounts of dried reagents (ng quantities and less) into microlitres of sample in a capillary-driven microfluidic chip. Typically, a RI is less than 1 mm(2) in area and has an inlet splitting into a central reagent channel, in which reagents can be loaded using an inkjet spotter, and two diluter channels. During filling of the microfluidic chip, spotted reagents reconstitute and exit the RI with a dilution factor that relates to the relative hydraulic resistance of the channels forming the RI. We exemplify the working principle of RIs by (i) distributing ～100 pg of horseradish peroxidase (HRP) in different volume fractions of a 1 μL solution containing a fluorogenic substrate for HRP and (ii) performing an immunoassay for C-reactive protein (CRP) using 450 pg of fluorescently labeled detection antibodies (dAbs) that reconstitute in ～5 to 30% of a 1 μL sample of human serum. RIs preserve the conceptual simplicity of lateral flow assays while providing a great degree of control over the integration and release of reagents in a stream of sample. We believe RIs to be broadly applicable to microfluidic devices as used for biological assays.     

Study of Interaction between Red-tide Toxin, Domoic Acid and Double ——stranded DNA by Capillary Zone Electrophoresis

The interactions between amnesic red-tide toxin, domoic acid (DA) and 14mer double-stranded DNA (dsDNA with three kinds of sequences) were studied by capillary zone electrophoresis (CZE). For the dsDNA with a sequence of 5‘-CCCCCTATACCCGC-3‘, the amount of free dsDNA decreases with the increase of added DA, and the signal of DA-dsDNA complex was observed. Meanwhile, the other two dsDNAs, 5‘-(C)12GC-3‘ and 5‘-(AT)7-3‘, the existence of DA could not lead to the change of dsDNA signal and indicated that there is no interaction between DA and these two dsDNAs.     

Influence of temperature and interactions with ligands on dissociation of dsDNA and ligand-dsDNA complexes of various types of binding. An electrochemical study

Several medicinally important compounds that bind to dsDNA strands via intercalation (C-1311, C-1305, EtBr), major groove binding (Hoechst 33258) and covalent binding (cis-Pt) were examined. The obtained results suggest that both the transfer of conformation B to C and the denaturation process, for the ligand-dsDNA complexes, except for covalently bound cis-Pt, took place at higher temperatures compared to the unbound helix. Furthermore, much lower currents of electrooxidation of guanine at 100 °C, compared to the currents obtained at this temperature for dsDNA in the absence of ligands, suggest that the binding of ligands affects the way the dsDNA denaturates at increased temperatures and leads to formation of different forms of DNA single strands. The voltammetric results were compared with the data of two spectroscopic techniques: UV-Vis and CD.