On-chip isoelectric focusing using photopolymerized immobilized pH gradients

We present the first successful adaptation of immobilized pH gradients (IPGs) to the microscale (muIPGs) using a new method for generating precisely defined polymer gradients on-chip. Gradients of monomer were established via diffusion along 6 mm flow-restricted channel segments. Precise control over boundary conditions and the resulting gradient is achieved by continuous flow of stock solutions through side channels flanking the gradient segment. Once the desired gradient is established, it is immobilized via photopolymerization. Precise gradient formation was verified with spatial and temporal detection of a fluorescent dye added to one of the flanking streams. Rapid (<20 min) isoelectric focusing of several fluorescent pI markers and proteins is demonstrated across pH 3.8-7.0 muIPGs using both denaturing and nondenaturing conditions, without the addition of carrier ampholytes. The muIPG format yields improved stability and comparable resolution to prominent on-chip IEF techniques. In addition to rapid, high-resolution separations, the reported muIPG format is amenable to multiplexed and multidimensional analysis via custom gradients as well as integration with other on-chip separation methods.     

Microfluidic digital isoelectric fractionation for rapid multidimensional glycoprotein analysis

Here we present an integrated microfluidic device for rapid and automated isolation and quantification of glycoprotein biomarkers directly from biological samples on a multidimensional analysis platform. In the first dimension, digital isoelectric fractionation (dIEF) uses discrete pH-specific membranes to separate proteins and their isoforms into precise bins in a highly flexible spatial arrangement on-chip. dIEF provides high sample preconcentration factors followed by immediate high-fidelity transfer of fractions for downstream analysis. We successfully fractionate isoforms of two potential glycoprotein cancer markers, fetuin and prostate-specific antigen (PSA), with 10 min run time, and results are compared qualitatively and quantitatively to conventional slab gel IEF. In the second dimension, functionalized monolithic columns are used to capture and detect targeted analytes from each fraction. We demonstrate rapid two-dimensional fractionation, immunocapture, and detection of C-reactive protein (CRP) spiked in human serum. This rapid, flexible, and automated approach is well-suited for glycoprotein biomarker research and verification studies and represents a practical avenue for glycoprotein isoform-based diagnostic testing.     

DNA extraction using a tetramethyl orthosilicate-grafted photopolymerized monolithic solid phase

A novel high-capacity, high-efficiency DNA extraction method is described using a photopolymerized silica-based monolithic column in a fused-silica capillary. Development involved investigation of the composition of the sol-gel monomer, fabrication conditions, and surface modifications in order to optimize the binding capacity. Extraction capacity and efficiency with the 3-(trimethoxysilyl)propyl methacrylate (TMSPM) monolith formulations fabricated in capillaries were investigated using a simple three-step procedure consisting of sample loading, washing of the solid phase, and elution of the DNA using a low ionic strength Tris buffer at pH 8. Once the TMSPM monomer concentration was optimized to yield a monolith with maximum test stability (robustness) and minimum back pressure, the monolith surface was modified by the grafting of tetramethyl orthosilicate (TMOS) for increased DNA binding capacity. After the examination of a variety of TMOS concentrations, 85% v/v TMOS was found to be optimal for DNA extraction without any obvious changes to the monolith structure. The reduction of time allowed for TMSPM hydrolysis prior to UV polymerization from 20 to 5 min led to a lower back pressure of the monolith, enabling better TMOS derivatization and therefore higher binding capacity. Minimal buffer volume (as low as 1 muL) was required to elute DNA from the solid phase, providing a DNA concentrating effect potentially important for downstream processes. While experimentation employed monolithic columns that were 12 cm in length, reduction of the length to 2 cm still allowed for a DNA binding capacity of at least 100 ng of prepurified human genomic DNA and extraction efficiencies greater than 85%. Extraction of low sample volumes (submicroliter) of human whole blood were successfully performed, with extraction efficiencies from the 2-cm monolithic column higher than those obtained from a commercial DNA extraction kit. These results position this novel matrix as an attractive alternative for solid-phase extraction of DNA and other biologically active molecules in microscale devices.     

Possibilities of using composite track membranes with nitride coating for fractionation of microelements in natural water

A plasma chemical procedure was developed for preparing composite track membranes with a conducting coating of titanium nitride particles having a complex hierarchic structure. Owing to oxynitride deposition on the surface of a poly(ethylene terephthalate) track membane, the membrane channels become asymmetrical and the membrane strength is enhanced. The pore opening size in composite membranes can be varied in a wide range by varying the synthesis parameters. The possibilities of directly using composite track membranes for electron-microscopic imaging of the form and composition of nonconducting mineral and biological substances and for qualitative and quantitative size fractionation of microelements in natural water by ultrafiltration were demonstrated by the example of a series of macro- (C, Al, Si, Mg, Fe, P) and microelements (Sr, U, Th).     

Studies of histidine as a suitable isoelectric buffer for tryptic digestion and isoelectric trapping fractionation followed by capillary electrophoresis-mass spectrometry for proteomic analysis

The use of histidine as a protein digestion buffer followed by isoelectric trapping separations using "membrane separated wells for isoelectric focusing and trapping" (MSWIFT) and mass spectrometry (MS) analysis is described. Tryptic digestion of bovine serum albumin (BSA) performed in histidine buffered solutions yields similar amino acid sequence coverage values to those obtained using ammonium bicarbonate buffer. Time course studies suggest that histidine buffers provide faster migration of peptides from the loading compartment compared to digestions prepared in ammonium bicarbonate due to differences in conductivities of the two buffers. In addition, this sample preparation method and MSWIFT separations have been coupled with capillary electrophoresis (CE) and matrix-assisted laser desorption ionization-mass spectrometry (MALDI-MS) as an alternative separation approach for proteomic studies. Tryptic peptides of ribosomal proteins in histidine are fractionated using MSWIFT followed by CE-MALDI-MS, which further illustrates the ability to couple fractions from a pI based separation device to CE-MS. Specifically, two-dimensional CE-MS plots provide a direct correlation between the numbers of basic residues within the peptide sequence displayed in charge-state trend lines. Combining MSWIFT and CE-MS provides added information regarding peptide sequence, specifically pI and in-solution charge state. Post-translational modifications can also be identified using this method.     

Selective enrichment and fractionation of phosphopeptides from peptide mixtures by isoelectric focusing after methyl esterification

We have developed a new strategy to enrich and fractionate phosphopeptides from peptide mixtures based on the difference in their isoelectric points (pIs) after methyl esterification. After isoelectric focusing (IEF) of a methylated tryptic digest of a mixture of alpha-S-casein and beta-casein, phosphopeptides were selectively enriched at acidic and neutral pHs while nonphosphopeptides left the focusing gel because their pIs are higher than the upper limit of the immobilized pH gradient. We wrote a web-based program, pIMethylation, to predict the pIs for peptides with and without methyl esterification. Theoretical calculations using pIMethylation indicated that methylated phosphopeptides and non-phosphopeptides can be grouped on the basis of the number of phosphate groups and basic residues in each peptide. Our IEF results were consistent with theoretical pIs of methylated peptides calculated by pIMethylation. We also showed that 2,6-dihydroxy-acetophenone is superior to 2,5-dihydroxybenzoic acid as a matrix for MALDI Q-TOF MS of methylated phosphopeptides in both positive and negative ion modes.     

Depth characterization of photopolymerized films by confocal Raman microscopy using an immersion objective

The depth characterization of photopolymer films by confocal Raman microscopy is often troublesome due to refraction effects. To minimize these effects, we used an oil immersion objective and a method was developed to avoid penetration of the oil without damaging the sample surface. Since the surface may be sticky if oxygen in the air inhibits the photopolymerization, a protective layer could not be put onto the film. Therefore, the method consists in using a thin polypropylene foil as substrate for the coating and placing the sample upside down under the objective. In this manner, the immersion oil could be deposited on top of the polypropylene. The advantage of this setup is that the oil, polypropylene substrate, and photopolymer film have close refractive indices. Basic calculations showed that the depth resolution is hardly affected in that configuration and double-bond conversion profiles could be plotted as a function of reliable nominal depth. The validity of the methodology was confirmed by experiments carried out with a dry metallurgical objective on the sample surface, face up, where refraction effects are still minor. In addition, infrared spectroscopy, which was used to follow the photopolymerization, corroborated the Raman conversion of the films over their thickness. The confocal Raman microscopy method can be applied to various photopolymerized systems to characterize their behavior towards oxygen inhibition and other heterogeneities in conversion arising from inner filter effects or interactions between additives for instance.     

Integration of dialysis membranes into a poly(dimethylsiloxane) microfluidic chip for isoelectric focusing of proteins using whole-channel imaging detection

A poly(dimethylsiloxane) microfluidic chip-based cartridge is developed and reported here for protein analysis using isoelectic focusing (IEF)-whole-channel imaging detection (WCID) technology. In this design, commercial dialysis membranes are integrated to separate electrolytes and samples and to reduce undesired pressure-driven flow. Fused-silica capillaries are also incorporated in this design for sample injection and channel surface preconditioning. This structure is equivalent to that of a commercial fused-silica capillary-based cartridge for adapting to an IEF analyzer (iCE280 analyzer) to perform IEF-WCID. The successful integration of dialysis membranes into a microfluidic chip significantly improves IEF repeatability by eliminating undesired pressure-driven hydrodynamics and also makes sample injection much easier than that using the first-generation chip as reported recently. In this study, two microfluidic chips with a 100-microm-high, 100-microm-wide and a 200-microm-high, 50-microm-wide microchannel, respectively, were applied for qualitative and quantitative analysis of proteins. The mixture containing six pI markers with a pH range of 3-10 was successfully separated using IEF-WCID. The pH gradient exhibited a good linearity by plotting the pI value versus peak position, and the correlation coefficient reached 0.9994 and 0.9995 separately for the two chips. The separation of more complicated human hemoglobin control sample containing HbA, HbF, HbS, and HbC was also achieved. Additionally, for the quantitative analysis, a good linearity of IEF peak value versus myoglobin concentration in the range of 20-100 microg/mL was obtained.     

Study of nanoscale photopolymerized fullerene clusters in solution droplets using an ultrasonic nebulizer unit

In a designed and developed ultrasonic nebulizer system for obtaining macroscopic-quantity photopolymerized fullerene (C60) clusters, a C60 solution was vaporized to several micro-sized droplets in vacuum, resulting in the formation of C60 aggregates by evaporating the solvent (toluene). The system was invented to produce nanoscale photopolymerized carbon clusters through the irradiation of ultraviolet (UV) light on the C60 aggregates in vacuum. The products, photopolymerized C60 clusters obtained from the system using UV-visible (UV-Vis) absorption and high-performance (or high-pressure) liquid chromatography (HPLC) spectra, were characterized. Compared with the non-irradiating C60 solution, the UV-Vis absorption spectrum of the irradiated C60 solution was drastically decreased, especially at lambda = 335 nm and in the visible region from lambda = 450-650 nm. As such, the UV-Vis absorption spectra provide information about the polymerization of C60 molecules. These photopolymerized C60 clusters can be detected as having a heavy molecular mass order through the HPLC system, and the C60 and photopolymerized C60 cluster can be extracted from the trapped solution on the molecular mass. Although there is a possibility that the products include various forms of C60 clusters, the results suggest that the products obtained from the system using a vaporizer establish a new method of obtaining macroscopic-quantity C60 clusters.     

Two-photon photopolymerized tips for adhesion-free scanning-probe microscopy

A hydrophobic polymeric tip of atomic force microscopy has been fabricated by two-photon adsorbed photopolymerization methods (TPAP). The fabrication was performed by a layer-by-layer polymerization of sliced multiple three-dimensional computer-aided design data. The used base resin was composed of acrylate and epoxy, which showed hydrophobic properties after the photopolymerization. For the sharp tip fabrication, we used a "dynamic partial polymerization method" which applied "the threshold effect" of TPAP to maximize the fabrication resolution. To investigate the performance of the fabricated polymeric tip, we have imaged several organic, inorganic, and biological samples using contact or dynamic force mode. The imaging results showed the hydrophobic polymeric tips solved various problems related to the tip adhesion to hydrophilic sample surfaces. Finally, the topographic image resolution of sub-5 nm was obtained using the polymeric tips for the hydrophilic mica surface.     

Gelation of photopolymerized hyaluronic acid grafted with glycidyl methacrylate

Experiments have tracked the ambient gelation of a series of hydrophilic hyaluronic acid (HA) resins grafted with glycidyl methacrylate (GM) and photopolymerized as a function of dose. The resin mixtures range in GMHA concentration between 0.5 and 1.5% w/w in phosphate buffered saline (PBS). Illuminated at 20 mW/cm², the dynamic viscosity (η(t)) has been tracked and characterized using the Boltzmann log-sigmoidal model. A gelled viscosity of ~ 10 Pa s was determined at 0.5% w/w which rose to ~ 50 Pa s at or above 1% w/w. More curing agent marginally increased the gel viscosity at each concentration. Time constants associated with viscosity advancement were shortest at [GMHA] = 1.0%; higher concentrations are attributed with lower quantum efficiency when illuminated. Subsequent frequency sweeps replicated already published work using similar GHMA concentrations in PBS. G′ values ranged from 100 to 500 Pa over the formulation range with expected sensitivity to GMHA and curing agent concentration. Overall, the sigmoidal model represented this advancing viscosity data well, and further analysis of the physical significance of these model parameters may help in understanding photopolymerization of this complicated formulation more broadly.     

Dynamic cell fractionation and transportation using moving dielectrophoresis

This study presents a new cell manipulation method using a moving dielectrophoretic force to transport or fractionate cells along a microfluidic channel. The proposed moving dielectrophoresis (mDEP) is generated by sequentially energizing a single electrode or an array of electrodes to form an electric field that moves cells continuously along the microchannel. Cell fractionation is controlled by the applied electrical frequency, and cell transportation is controlled by the interelectrode activation time. The applicability of this method was demonstrated to simultaneously fractionate and transport Saccharomyces cerevisiae yeast cells, both viable and nonviable, by operating at conditions under which the cells were subjected to positive and negative dielectrophoresis, respectively. Compared to the conventional dielectrophoresis (cDEP and traveling wave dielectrophoresis (twDEP), moving dielectrophoresis allows cells to be separated on the basis of the real part of the Clausius-Mossotti factor, as in cDEP, but yet allows the direct transportation of separated cells without using fluid flow, as in twDEP. This dielectrophoresis technique provides a new way to manipulate cells and can be readily implemented on programmable multielectrode devices.     

Field-effect detection using phospholipid membranes

The application of field-effect devices to biosensors has become an area of intense research interest. An attractive feature of field-effect sensing is that the binding or reaction of biomolecules can be directly detected from a change in electrical signals. The integration of such field-effect devices into cell membrane mimics may lead to the development of biosensors useful in clinical and biotechnological applications. This review summarizes recent studies on the fabrication and characterization of field-effect devices incorporating model membranes. The incorporation of black lipid membranes and supported lipid monolayers and bilayers into semiconductor devices is described.     

Field-effect detection using phospholipid membranes

Abstract

The application of field-effect devices to biosensors has become an area of intense research interest. An attractive feature of field-effect sensing is that the binding or reaction of biomolecules can be directly detected from a change in electrical signals. The integration of such field-effect devices into cell membrane mimics may lead to the development of biosensors useful in clinical and biotechnological applications. This review summarizes recent studies on the fabrication and characterization of field-effect devices incorporating model membranes. The incorporation of black lipid membranes and supported lipid monolayers and bilayers into semiconductor devices is described.     

Single-molecule spectroscopy using nanoporous membranes

We describe a novel approach for optically detecting DNA translocation events through an array of solid-state nanopores that potentially allows for ultra high-throughput, parallel detection at the single-molecule level. The approach functions by electrokinetically driving DNA strands through sub micrometer-sized holes on an aluminum/silicon nitride membrane. During the translocation process, the molecules are confined to the walls of the nanofluidic channels, allowing 100% detection efficiency. Importantly, the opaque aluminum layer acts as an optical barrier between the illuminated region and the analyte reservoir. In these conditions, high-contrast imaging of single-molecule events can be performed. To demonstrate the efficiency of the approach, a 10 pM fluorescently labeled lambda-DNA solution was used as a model system to detect simultaneous translocation events using electron multiplying CCD imaging. Single-pore translocation events are also successfully detected using single-point confocal spectroscopy.     

Structural analysis on photopolymerized dental resins containing nanocomponents

The incorporation of nanoscale layered silicates, as montmorillonite (MMT), into polymers has attracted great attention due to their ability to improve mechanical, thermal, and barrier properties of many polymers. Inserting these nanocomponents into dental resins could lead to materials with improved wearing resistance under masticatory attrition, among other advantages. Thus, the aim of this work was to investigate the process of incorporating layered silicates into dental resins by studying the influence of the concentration of these nanocomponents and the type of chemical species, used to modify the clays, in the morphology of the systems. Three types of clay were inserted in a BisGMA/TEGDMA photopolymerized resin system. The samples were characterized by scanning electron microscopy (SEM), X-ray diffraction (XRD), thermogravimetric analysis (TGA), infrared spectroscopy (FTIR) and small angle X-ray scattering (SAXS). FTIR and TGA results confirmed the presence of the modifier agents in MMT. SEM micrographs indicated a homogeneous distribution of the layered silicates throughout the dental resin after photopolymerization and a high degree of interfacial adhesion. SEM micrographs also showed that the mixing process used to add clay into dental monomers was not successful in breaking agglomerates and was also responsible for forming new agglomerates particularly in highly concentrated materials. XRD and SAXS patterns showed the development of a partially exfoliated/intercalated clay structure after photopolymerization of the dental resin. Exfoliation, together with intercalation, was even observed in materials having concentrations of MMT as high as 16 wt.%. Exfoliation in highly filled materials was attributed to the formation of a microstructure composed of large agglomerates, in which most of the MMT layers were concentrated and restricted to exfoliate, and few small particles disseminated throughout the polymer matrix that were able to exfoliate. SAXS results also suggested that the modification of MMT with species containing polar groups was more successful in producing exfoliated structures than MMT having highly hydrophobic incorporated species. The insertion of vinyl groups into MMT galleries led to the production of dental resins containing large and compact agglomerates with intercalated layers, as suggested by determining the fractal number of the materials. The possibility of producing photopolymerized dental resins containing exfoliated nanolayers can potentially be useful in controlling important properties of dental materials such as resistance to attrition, moisture absorption, polymerization shrinkage, coefficient of thermal expansion, among others.