Microfluidic self-assembly of insulin monomers into amyloid fibrils on a solid surface

We report the self-assembly of insulin monomers into amyloid fibrils within microchannels. To demonstrate the microfluidic amyloid formation and fibril growth on a solid surface, we seeded the internal surfaces of the microchannels with insulin monomers via N-hydroxysuccinimide ester activation and continuously flushed a fresh insulin solution through the microchannels. According to our analysis using optical and fluorescence microscopy, insulin amyloid preferentially formed in the center of the microchannels and, after reaching a certain density, spread to the side walls of the microchannels. By using ex situ atomic force microscopy, we observed the growth of amyloid fibrils inside the microchannels, which occurred at a much higher rate than that in bulk systems. After 12 h of incubation, insulin formed amyloid spherulites having "Maltese cross" extinction patterns within the microchannels according to the polarized microscopic analysis. Microfluidic amyloid formation enabled low consumption of reagents, reduction of incubation time, and simultaneous observation of amyloid formation under different conditions. This work will contribute to the rapid analysis of amyloid formation associated with many protein misfolding diseases.     

Two-dimensional ordered arrays of aligned lipid tubules on substrates with microfluidic networks

Microfluidic networks is a powerful tool for aligning one-dimensional materials over a large area on solid substrates. Here we show that lipid nano- and microtubules can be assembled into two-dimensional (2-D) parallel arrays with controlled separations by combining fluidic alignment with dewetting, which occurs within microchannels. We also demonstrate that lipid tubules can be bent into a well-defined shape at the entrance of the channels by the capillary force. Atomic force microscopy is used to study the structure and stability of the aligned lipid tubules on substrates. The deposition experiments with silica colloidal particles show that the 2-D parallel-aligned tubules can be used as a template to synthesize silica films with controlled morphologies and patterns on substrates in a single-step process.     

Microchannels filled with diverse micro- and nanostructures fabricated by glancing angle deposition

The integration of porous structures into microchannels is known to enable unique and useful separations both in electrophoresis and chromatography. Etched pillars and other nanostructures have received considerable interest in recent years as a platform for creating microchannels with pores tailored to specific applications. We present a versatile method for integration of three-dimensionally sculptured nano- and micro-structures into PDMS microchannels. Glancing angle deposition was used to fabricate nanostructures that were subsequently embedded in PDMS microchannels using a sacrificial resist process. With this technique, an assortment of structures made from a wide selection of materials can be integrated in PDMS microchannels; some examples of this versatility, including chiral and chevron nanostructures, are demonstrated. We also present a working device made using this process, separating 6/10/20 kbp and 10/48 kbp DNA mixtures in a DNA fractionator containing GLAD-deposited SiO(2) vertical posts as the separating medium. The separation mechanism was verified to resemble that found in prior fractionation devices, using total internal reflection fluorescence microscopy. GLAD fabrication enables insertion of three-dimensional structures into microchannels that cannot be fabricated with any existing techniques, and this versatility in structural design could facilitate new developments in on-chip separations.     

Inkjet printing of laminin gradient to investigate endothelial cellular alignment

To investigate the influence of the protein surface-density gradient on endothelial cell alignment, a novel approach for the fabrication of a laminin gradient on gold-coated substrates has been developed in this study. Our approach involves programmed inkjet printing of an alkanethiol (11-mercaptoundecanoic acid, C₁₀COOH, MUA) gradient onto gold-coated substrates, followed by backfilling with 11-mercapto-1-undecanol (C₁₁OH, MUD). The –COOH moieties were activated and then covalently linked with laminin. This treatment led to a surface-density gradient of laminin. Contact angle measurement, X-ray photoelectron spectroscopy (XPS) and fluorescence microscopy were employed to characterize the self-assembled monolayers (SAMs) and protein gradient, respectively. Results proved the feasibility of the fabrication of a protein gradient by using the inkjet printing technique. The self-assembled monolayer gradients displayed a high packing density, as indicated by dynamic contact angle measurement. More importantly, the gradient slope was easily tunable over a significant distance from 20 to 30 mm. The laminin gradient was clearly visible by fluorescence microscopy observation. Endothelial cells cultured on the surface-density gradient of laminin demonstrated a strong alignment tendency in parallel to the gradient. The higher the laminin density the more cells were observed. The result indicates that cell attachment is dependent on the surface density of laminin. This work broadens our methodology to investigate chemical stimuli-induced cell directional alignment. It is potentially important for understanding cell alignment/ingrowth behavior for angiogenesis and implant technology including tissue-engineered structures.     

Differential interference contrast microscopy for real-time dynamics and manipulation of single cells in microchannels

Nomarski differential interference contrast (DIC) microscopy was used for real-time dynamics of intact single cells in various microchannels for adaptation to microfluidic chip application. The cheek cell was chosen as a model, single cell and the dynamics was measured at the microchannels. The image resolution of single cell was shaper and more distinct in DIC than in conventional microscopy. The individual single living cells were also manipulated by both hydrodynamic and electrokinetic flow-driving forces at the microchannels. The DIC contrast was enhanced according to the order of round-, square-, and rectangle-type microchannels. The velocity of the single living cell was consistently increased with increasing electric field strength and pH. However, the velocity of cell was decreased with increasing run buffer concentration. The driving direction of the individual single cell was simply controlled by changing the polarity of the applied voltage and the electric field strength. The cells were consistently manipulated in the microchannel under the co-application of the low electric field of 2.44 V/cm, instead of the solo application of the hydrodynamic force.     

Characterization of molecular transport in poly(dimethylsiloxane) microchannels for electrophoresis fabricated with synchrotron radiation-lithography and UV-photolithography

In the present paper, a study was undertaken of molecular transport in ploy(dimethylsiloxane) microchannels that were fabricated by ultraviolet (UV)-photolithography and synchrotron radiation (SR)-lithography characterized and compared for microchip capillary electrophoresis by evaluating in-channel molecular dispersion. A fluorescent tag, sulforhodamine B was used as the probing molecule. It was found that microchannels made by SR-lithography fabrication were superior to those made by UV-photolithography fabrication in terms of molecular transport performance. A deep insight into surface conditions characterized by scanning electron microscopy suggested it was related to the difference in surface roughness. Chromatographic retention in electropherograms further supported such a conclusion, which depended on the phase ratio of the channel surface. The results revealed for PDMS microchannels in this work were in good agreement with the phenomenon found for glass microchannels in the literature.     

Rigid, superparamagnetic chains of permanently linked beads coated with magnetic nanoparticles. Synthesis and rotational dynamics under applied magnetic fields

An inexpensive and versatile approach is reported for the synthesis of monodisperse magnetoresponsive rods of desired diameter, length, and magnetic susceptibility based on the confined alignment of magnetic beads in microchannels of selected channel height, followed by localized hydrolysis of sol-gel precursors within polyelectrolyte shells adsorbed on the beads. The layer-by-layer technique was used to coat the polystyrene beads with polyelectrolytes of alternating charge and with charged magnetic nanoparticles, and the polystyrene cores could be removed either by solvent dissolution or by calcination to form hollow-shelled chains. The reorientation dynamics of single and clustered chains following the application of an external magnetic field was evaluated theoretically, with favorable comparisons with the experimental data.     

Fabrication of PDMS/glass microchips by twofold replication of PDMS and its application in genetic analysis

In this paper, we describe a simple method for fabrication of high quality poly(dimethylsiloxane) (PDMS)/glass microchip by twofold replica molding of PDMS. This technique first served to transfer the negative microchannels from the glass template to the PDMS substrate as a master, and then this PDMS master with positive microchannels was used to replicate the PDMS replica with negative microchannels. Finally, the PDMS replica was bound to a glass sheet by UV radiation. The fabricated microchips were successfully applied for the detection of C677T mutation from the human methylenetetrahydrofolate reductase gene.     

Fabrication of carbon microelectrodes with a micromolding technique and their use in microchip-based flow analyses

In this paper, we report a new technique to pattern carbon microelectrodes for use in microfluidics. This technique, termed micromolding of carbon inks, uses poly(dimethylsiloxane)(PDMS) microchannels to define the size of the microelectrode. First, PDMS microchannels of the approximate dimensions desired for the microelectrode are made by soft lithography. The PDMS is then reversibly sealed to a substrate and the microchannels are filled with carbon ink. After a heating step the PDMS mold is removed, leaving a carbon microelectrode with a size slightly smaller than the original PDMS microchannel. The resulting microelectrode (27 microm wide and 6 microm in height) can be reversibly sealed to a PDMS-based flow channel. Fluorescence microscopy showed that no leakage occurred around the chip/electrode seal, even up to flow rates of 10 microL min(-1). The electrode was characterized by microchip-based flow injection analysis. Injections of catechol in Hank's Balanced Salt Solution (pH 7.4), showed a linear response from 2 mM to 10 microM (r(2)= 0.995), with a sensitivity of 56.5 pA microM(-1) and an estimated limit of detection of 2 microM (0.27 picomole, S/N=3). Reproducibility of the electrode response was shown by repeated injections (n= 10) of a 500 microM catechol solution, resulting in a RSD of 4.6%. Finally, selectivity was demonstrated by coating the microelectrode with Nafion, a perfluoronated cation exchange polymer. Dopamine exhibited a response at the modified microelectrode while ascorbic acid was rejected by the Nafion-coating. These electrodes provide inexpensive detectors for microfluidic applications while also being viable alternatives to use of other carbon microelectrode materials, such as carbon fibers. Furthermore, the manner in which the microelectrodes are produced will be of interest to researchers who do not have access to state of the art microfabrication facilities.     

Multitrack electrospray chips

Multitrack electrospray chips (MTEC) were fabricated by UV-photoablation of polyethylene terephthalate (PET) substrates. They are composed of an array of up to six microchannels that are successively used as electrospray ionization (ESI) emitters for mass spectrometry (MS). There is no requirement for alignment of the different spraying microchannels with the mass spectrometer orifice. The MTEC is thus fixed in front of the mass spectrometer and the successive MS analyses are performed without moving the chip. The sequential electrospraying by successive application of an identical high voltage in each off-axis microchannel was evaluated for the fast screening of peptides and proteins. The counting of cysteines in peptides through chemical modification and the relative quantification of a peptide in two samples are presented herein as two original strategies based on this new analytical tool.     

Control of liquid crystal pretilt angles by chemical derivatization reaction of polyvinyl alcohol films

A chemical derivatization technique was used to control the pretilt angle of a liquid crystal. A polyvinyl alcohol (PVA) alignment layer, which gives a very low pretilt angle when in contact with the liquid crystal (LC), was reacted with trifluoroacetic anhydride (TFAA) in the gas phase to change polar -OH groups to -OCOCF₃ groups. By introduction of the -OCOCF₃ groups in to the PVA, we obtained homeotropic alignment of the E7 LC molecules. The homeotropic alignment of E7 LC molecules in contact with the derivatized PVA alignment layer was confirmed by FTIR and microscopy with crossed polarizers. The change of liquid crystal molecules from homogeneous to homeotropic alignment may be caused by the decrease in surface tension of the PVA alignment layer, due to substitution of the polar -OH groups by -OCOCF₃ groups in the gas phase derivatization reaction.     

Different types of electrospun nanofibers and their effect on microfluidic‐based immunoassay

Protein capturing on polymeric substrate of microfluidic devices is a key factor for the fabrication of immunoassay with high sensitivity. In this work, simple and versatile technique of electrospinning was used to produce electrospun nanofibrous membranes (e.NFMs) with high surface area as a substrate for microfluidic‐based immunoassay to increase sensitivity. It was found that the simultaneous use of e.NFM and 1‐Ethylethyl‐3‐(3‐dimethylaminopropyl)‐carbodiimide/N‐Hydroxysuccinimide hydroxysuccinimide as coupling agent has synergic effect on antigen immobilization onto the microchannels. It was found that the oxygen plasma technique for the creation of oxygen containing functional group like carboxyl and hydroxyl causes extreme leakage of solution through the microchannels. Thus, due to capillary effect, it is impossible to use hydrophilic substrate to modify microchannels. In order to compensate this problem, it is propose to utilize other type of polymer for the fabrication of nanofiber to answer this important question that if it is possible to enhance the sensitivity of immunoassay just by changing the polymer type? For this purpose, four different polymers, namely, polycaprolactone, poly lactic‐co‐glycolic acid, poly L‐lactic acid, and polyethersolfone were used as the based material for e.NFM fabrication. Results showed that compared with plain poly (dimethylsiloxane) surface of microchannels, poly lactic‐co‐glycolic acidand poly L‐lactic acid, which inherently contain end‐group of carboxyl in their chemical structure, can improve the protein immobilization, which leads to immunoassay signal enhancement through 1‐ethyl‐3‐(3‐dimethylaminopropyl)‐carbodiimide/N‐hydroxysuccinimide coupling chemistry, significantly.     

Fluidic assembly and packing of microspheres in confined channels

We study fluidic assembly and packing of spherical particles in rectilinear microchannels that are terminated by a flow constriction. First, we introduce a method for active assembly of particles in the confined microchannels by triggering a local constriction in the fluid channel using a partially closed membrane valve. This microfluidic valve allows active, on-demand particle assembly as opposed to previous passive assembly methods based on terminal channels and weirs. Second, we study the three-dimensional assembly and packing of particles against a weir in confined rectilinear microchannels. The packings result in achiral particle chains with alternating (zigzag) structure. This structure is characterized by a single, repeated bond angle whose components projected into the frame of the channel are quantified by confocal microscopy and image processing. Brownian dynamics simulation of the packing comprehensively delineates the range of bond angles possible in narrow, rectilinear microchannels as well as the complex dependence of these angles on the relative dimensions of the channel and particles. The simulations of the three-dimensional packings are accurately modeled by a compact theory based on trigonometric relationships. The experimentally measured bond angles show excellent agreement with the simulations, thereby validating the functional dependence of the achiral packing bond angles on channel dimensions. This functional relationship is immediately useful for the design of anisotropic particles by microfluidic synthesis.     

On-line coupling of micro-enzyme reactor with micro-membrane chromatography for protein digestion, peptide separation, and protein identification using electrospray ionization mass spectrometry

To miniaturize high-performance membrane chromatography, a poly(vinylidene fluoride) membrane medium, employed as the stationary phase, is sandwiched between two poly(dimethylsiloxane) substrates containing the microchannels. The microchannels are fabricated by the capillary molding technique, involving the use of capillaries as the channel template and the fluid inlet/outlet. The micro(micro)-membrane chromatography system is coupled with a micro-enzyme reactor containing immobilized trypsins for performing rapid protein digestion, peptide separation, and protein identification using electrospray ionization mass spectrometry. Separation performance of cytochrome c digest in micro-membrane chromatography is compared with the results obtained from a regular reversed-phase micro-liquid chromatography. The efficacy and the potentials of micro-membrane chromatography in tryptic mapping are reported. On-line integration of the micro-enzyme reactor with micro-chromatographic separation techniques and electrospray ionization mass spectrometry clearly provides a microanalytical platform for automated sample handling, minimized sample loss, and reduced sample consumption. It also provides enhanced detection sensitivity and dynamic range for the analysis of complex protein mixtures such as cell lysates in proteomics research.     

Real-time assessment of carbon nanotube alignment in a polymer matrix under an applied electric field via polarized Raman spectroscopy

A technique has been developed utilizing polarized Raman spectroscopy to measure alignment of carbon nanotubes in situ in a polymer matrix under an applied electric field. Previous studies of alignment have been restricted to optically transparent solvents or polymerized specimens that prevent accurate analyses of alignment dynamics in polymers. The effects of electric field strength on the degree of alignment and the time to achieve an aligned state are discussed. The use of in situ, real-time polarized Raman spectroscopy provides a non-invasive technique for assessing carbon nanotube alignment, which can assist in determining processing conditions to improve the mechanical and electrical properties of aligned nanocomposites.     

Microchannel systems in titanium and silicon for structural and mechanical studies of aligned protein self-assemblies

We report a technique for the alignment of self-assembled protein systems, such as F-actin bundles and microtubules, in a surface-modified titanium or silicon microfluidic device. Assembling filamentous protein systems in a confined geometry produces highly aligned samples for structural and mechanical studies. Biomolecular self-assembly can be investigated in a controlled fashion under different molecular concentration gradients and conditions along a channel length. We have shown that surface-modified devices produced via a high aspect ratio etch process in titanium and silicon can be used to confine and control such macromolecular assemblies and present examples of F-actin bundles and microtubules in this system.     

Control of liquid crystal pretilt angles by chemical derivatization reaction of polyvinyl alcohol films

A chemical derivatization technique was used to control the pretilt angle of a liquid crystal. A polyvinyl alcohol (PVA) alignment layer, which gives a very low pretilt angle when in contact with the liquid crystal (LC), was reacted with trifluoroacetic anhydride (TFAA) in the gas phase to change polar –OH groups to –OCOCF₃ groups. By introduction of the –OCOCF₃ groups in to the PVA, we obtained homeotropic alignment of the E7 LC molecules. The homeotropic alignment of E7 LC molecules in contact with the derivatized PVA alignment layer was confirmed by FTIR and microscopy with crossed polarizers. The change of liquid crystal molecules from homogeneous to homeotropic alignment may be caused by the decrease in surface tension of the PVA alignment layer, due to substitution of the polar –OH groups by –OCOCF₃ groups in the gas phase derivatization reaction.     

Programmable modification of cell adhesion and zeta potential in silica microchips

Spatial patterning of thin polyacrylamide films bonded to self-assembled monolayers on silica microchannels is described as a means for manipulating cell-adhesion and electroosmotic properties in microchips. Streaming potential measurements indicate that the zeta potential is reduced by at least two orders of magnitude at biological pH, and the adhesion of several kinds of cells is reduced by 80-100%. Results are shown for cover slides and in wet-etched silica microchannels. Because the polyacrylamide film is thin and transparent, this film is consistent with optical manipulation of cells and detection of cell contents. The spatial patterning technique is straightforward and has the potential to aid on-chip analysis of single adherent cells.     

Three-dimensional closed microfluidic channel fabrication by stepper projection single step lithography: the diabolo effect

Microfluidic devices are currently being used in many types of biochemical microsystems for liquid phase analysis in the frame of medical applications. This paper presents a new technique for the realization of microfluidic channels using SU-8, a commonly used epoxy-based negative photo-resist. These microchannels were fabricated by a single stepper UV-photolithography process. By changing the process parameters, e.g. the optical focus depth and the UV exposure dose, well-defined, covered microchannels with various dimensions and aspect ratios were realized and proven to be effective for the fluid transport by capillarity. This technique can easily be used for the fabrication of microfluidic devices in the microanalysis and lab-on-chip applications realm.     

Fabrication and characterization of poly(methyl methacrylate) microchannels by in situ polymerization with a novel metal template

A stainless steel template for the fabrication of plastic microfluidic devices has been developed by photolithography and chemical etching technique. The preparation process of the template is simple, rapid, and low-cost. The cross sectional profiles of raised microchannels on the template are trapezoidal. The surface roughness of the templates was controlled down to 190 nm. The template can be used repeatedly to generate devices reproducibly. The microfluidic devices of poly(methyl methacrylate) (PMMA) were fabricated by in situ polymerization using the templates. The reproducibility of the fabricated microchannel is high and the relative standard deviation is 0.7% by the in situ polymerization approach. Some physical properties of the polymerized microchannels were characterized including the transparency, the thermal deformation temperature, and the dimensional information. Current monitoring was used to evaluate the electroosmotic flow within the microchannels under the electric field strength of 300 V/cm.