Selective protein removal and desalting using microchip CE

This paper describes the on-line sample pretreatment and analysis of proteins and peptides with a poly(methylmethacrylate) (PMMA) microfluidic device (IonChip). This chip consists of two hyphenated electrophoresis channels with integrated conductivity detectors. The first channel can be used for sample preconcentration and sample clean-up, while in the second channel the selected compounds are separated. Isotachophoresis (ITP) combined with zone electrophoresis (CZE) was used to preconcentrate a myoglobin sample by a factor of about 65 before injection into the second dimension and to desalt a mixture of six proteins with 100 mM NaCl. However, ITP-CZE could not be used for the removal of two proteins from a protein/peptide sample since the protein zone in the ITP step was too small to remove certain compounds. Therefore, we used CZE-CZE for the removal of proteins from a protein/peptide mixture, thereby injecting only the peptides into the second CZE separation channel.     

Disposable polydimethylsiloxane/silicon hybrid chips for protein detection

This paper presents disposable protein analysis chips with single- or four-chamber-constructed from poly(dimethylsiloxane) (PDMS) and silicon. The chips are composed of a multilayer stack of PDMS layers that sandwich a silicon microchip. This inner silicon chip features an etched array of micro-cavities hosting polymeric beads. The sample is introduced into the fluid network through the top PDMS layer, where it is directed to the bead chamber. After reaction of the analyte with the probe beads, the signal generated on the beads is captured with a CCD camera, digitally processed, and analyzed. An established bead-based fluorescent assay for C-reactive protein (CRP) was used here to characterize these hybrid chips. The detection limit of the single-chamber protein chip was found to be 1 ng/ml. Additionally, using a back pressure compensation method, the signals from each chamber of the four-chamber chip were found to fall within 10% of each other.     

Development of low-cost microfluidic systems for lab-on-a-chip biosensor applications

In this work, we develop low-cost microfluidic systems based on polydimethylsiloxane (PDMS) for lab-on-a-chip applications. PDMS microfluidic structures have been fabricated by micromolding, PDMS casting, and plasma bonding processes. The micromolding technique is used to fabricate PDMS slabs with micro-sized grooves, and the complete microchannel is formed by bonding PDMS slab with glass or PDMS substrate. The molding procedure using SU-8 photoresist patterning on silicon wafer, PDMS microchannel fabrication, and PDMS surface treatment using oxygen plasma and TiO₂ coating, are discussed. The various parameters for oxygen plasma treatment including RF power and treatment time are studied in order to obtain conditions for good bonding with the glass substrate. The best condition for plasma treatment is found to be the low RF power (8 W) with 5 min treatment time. In addition, TiO₂ coating with oxygen plasma treatment has been applied to make PDMS surface more hydrophilic to improve aqueous solution compatilbility. The microfluidic channels for various applications, including sample injection cross channel, micropump channel, T and Y sample mixers, PCR thermocyling chamber and channel, capillary electrophoresis flow channel, and conductimetric systems have been fabricated. Finally, a typical application of the PDMS chip in a flow injection conductimetric system for sodium chloride detection has been demonstrated.     

Degassing‐assisted patterning of cell culture surfaces

We developed an alternative patterning technique which is capable of producing both topographic and biochemical features for cell culture studies. This technique is based on microaspiration induced with a degassed poly (dimethylsiloxane) (PDMS) mold. After degassing in a rough vacuum chamber and placed on a sample surface, liquid solution can be aspired through channels and cavities created in the PDMS mold. Depending on the composition of the solution and the associated drying or incubation processes, a variety of surface patterns can be produced without applying external pressure. For demonstration, we fabricated agarose gel microwells and biomolecule patterns either on a glass plate or in a cell culture Petri dish, both applicable for cell culture studies. Biotechnol. Bioeng. 2010. 105: 854–859. © 2009 Wiley Periodicals, Inc.     

ProteoChip: a highly sensitive protein microarray prepared by a novel method of protein immobilization for application of protein-protein interaction studies

We have developed a highly sensitive microarray protein chip, ProteoChip, coated with ProLinker, novel calixcrown derivatives with a bifunctional coupling property that permits efficient immobilization of capture proteins on solid matrixes and makes high-throughput analysis of protein-protein interactions possible. The analysis of quartz crystal microbalance showed that both monoclonal antibody (mAb) and antigen (Ag) bound to the gold film of the sensor surface coated with ProLinker B and that it is useful for studies of Ab-Ag interactions. ProteoChip, aminated glass slide coated with ProLinker A, was also demonstrated to be useful for preparation of high-density array spots by using a microarrayer and for analysis of analyte Ags either by direct or sandwich methods of fluorescence immunoassay. The detection sensitivity of ProteoChip was as low as 1-10 femtogram/mL of analyte protein, useful for detection of tumor markers. ProteoChip was also useful for studies of direct protein-protein interactions as demonstrated by analysis of integrin-extracellular matrix protein interaction. These experimental results suggest that ProteoChip is a powerful tool for development of chip-based lead screening microarrays to monitor protein-protein interactions (i.e. drug target) as well as for biomarker assays which require high detection sensitivity.     

Application of on-chip cell cultures for the detection of allergic response

In this report, the development of a microfluidic cell chip for monitoring allergic response is described. A rat basophilic leukemia cell line (RBL-2H3), a tumor analog of rat mucosal mast cells, has been used as a model to observe its allergic response upon antigen stimulus. The cells were cultivated on a poly(dimethylsiloxane) (PDMS) chip, the surface of which was modified by several methods. The PDMS chip, which comprised a cell cultivation chamber and microfluidic channels, was fabricated by conventional molding methods. In order to detect the allergic response, a fluorescent dye, quinacrine, was introduced inside the cell compartment that included histamine. The cells were stimulated with dinitrophenylated bovine serum albumin (DNP-BSA) after incubation with anti-DNP IgE. When exocytosis events occurred, the microfluidic system detected the fluorescent signal of quinacrine, which was released from RBL-2H3 cells by using a photomultiplier tube (PMT) fitted onto a microscope.     

Application of an enzyme chip to the microquantification of l-phenylalanine

We describe here a new microquantification method of l-phenylalanine concentration in an extract from a dried blood spot by using the diaphorase-resazurin system. To miniaturize the fluorometric enzymatic microplate assay for the diagnosis of phenylketonuria, an enzyme chip immobilized with His-tag fused phenylalanine dehydrogenase (PheDH) was developed. His-tag fused PheDH was immobilized on the surface of nickel-coated slide glass. A microarray sheet (8 x 30 well) was fabricated with poly(dimethylsiloxane) (PDMS) using the photolithographic technique. An enzyme reaction chamber in a double-layered structure was constructed with different types of microarray PDMS sheets on the surface of Ni-coated slide glass immobilized with His-tagged PheDH. To evaluate the affinity toward the Ni-chelating ligand, eight kinds of His-tagged PheDH variants were constructed and expressed. (His)(6)- and (His)(9)-PheDH variants at the N terminus showed high adsorption ratio to Ni-chelating ligand. The V(max) and k(cat) values of the (His)(6)-PheDH variant at the N terminus for l-phenylalanine were higher than those of the (His)(9)-PheDH variant, and the (His)(6)-PheDH variant was found to be most suitable for immobilization onto nickel-coated slide glass. Fluorescence formed by resazurin-coupled enzymatic reaction (in a 0.2-microl reaction mixture) on the enzyme chip exhibited good linearity and a correlation coefficient up to 12.8 mg/dl of the l-phenylalanine-containing sample extracted from a dried blood spot on filter paper.     

Application of immuno-mass spectrometry to analysis of a bacterial virulence factor

Here we describe a novel antibody-based assay that combines specificity of antibody with precision of mass spectral analysis. The assay is carried out in three steps using a single antigen capture and transfer reagent. The first step of the assay involves antibody immobilization. The second step is antigen capture and washing to remove unbound proteins. The third step involves the analysis of the captured antigens by surface enhanced laser desorption ionization time-of-flight mass spectrometry. The assay is facilitated by the ability of a single nonviable bacterial preparation expressing immunoglobulin-binding proteins that enable antibody immobilization, specific capture of fluid-phase antigen, and direct sample transfer to a protein chip for mass spectral analysis. Proof-of-concept studies using a model Streptococcus pyogenes virulence factor, the secreted cysteine protease SpeB, are presented.     

Immobilization of Piromyces rhizinflata β-Glucanase on Poly(Dimethylsiloxane) and Si Wafer and Prediction of Optimum Reaction for Enzyme Activity

EglA, a β-1,4-glucanase isolated from the ruminal fungus Piromyces rhizinflata, shows promise in a wide range of industrial applications because of its broad substrate specificity. In this study, EglA was immobilized on different supporting materials including poly(dimethylsiloxane) (PDMS), Si wafer, textured Si wafer, and indium tin oxide-coated (ITO-coated) glass. The binding abilities of PDMS and Si wafer toward EglA were significantly higher than those of the other supporting materials. The optimized temperature and pH conditions for EglA immobilized on PDMS and on Si wafer were further determined by a response surface methodology (RSM) combined with a central composite design (CCD). The results indicated that the optimum pH and temperature values as well as the specific β-glucanase activity of EglA on PDMS were higher than those of free-form EglA. In addition, EglA immobilized on PDMS could be reused up to six times with detectable enzyme activity, while the enzyme activity of Eg1A on Si wafer was undetectable after three cycles of enzyme reaction. The results demonstrate that PDMS is an attractive supporting material for EglA immobilization and could be developed into an enzyme chip or enzyme tube for potential industrial applications.     

Monosize poly(glycidyl methacrylate) beads for dye-affinity purification of lysozyme

Cibacron Blue F3GA was covalently attached onto monosize poly(glycidyl methacrylate) [poly(GMA)] beads for purification of lysozyme from chicken egg white. Monosize poly(GMA) beads, 1.6 microm in diameter, were produced by a dispersion polymerization technique. The content of epoxy groups on the surface of the poly(GMA) sample determined by the HCl-pyridine method (3.8 mmol/g). Cibacron Blue F3GA loading was 1.73 mmol/g. The monosize beads were characterized by elemental analysis, FTIR and SEM. Adsorption studies were performed under different conditions in a batch system (i.e., medium pH, protein concentration, temperature and ionic strength). Maximum lysozyme adsorption amount of poly(GMA) and poly(GMA)-Cibacron Blue F3GA beads were 1.6 and 591.7 mg/g, respectively. The applicability of two kinetic models including pseudo-first order and pseudo-second order model was estimated on the basis of comparative analysis of the corresponding rate parameters, equilibrium adsorption capacity and correlation coefficients. Results suggest that chemisorption processes could be the rate-limiting step in the adsorption process. It was observed that after 10 adsorption-elution cycle, poly(GMA)-Cibacron Blue F3GA beads can be used without significant loss in lysozyme adsorption capacity. Purification of lysozyme from egg-white was also investigated. Purification of lysozyme was monitored by determining the lysozyme activity using Micrococcus lysodeikticus as substrate. The purity of the eluted lysozyme was analyzed by SDS-PAGE and found to be 88% with recovery about 79%. The specific activity of the eluted lysozyme was high as 43,600 U/mg.     

Microfabricated polymer chip for capillary gel electrophoresis

A polymer (PDMS: poly(dimethylsiloxane)) microchip for capillary gel electrophoresis that can separate different sizes of DNA molecules in a small experimental scale is presented. This microchip can be easily produced by a simple PDMS molding method against a microfabricated master without the use of elaborate bonding processes. This PDMS microchip could be used as a single use device unlike conventional microchips made of glass, quartz or silicon. The capillary channel on the chip was partially filled with agarose gel that can enhance separation resolution of different sizes of DNA molecules and can shorten the channel length required for the separation of the sample compared to capillary electrophoresis in free-flow or polymer solution format. We discuss the optimal conditions for the gel preparation that could be used in the microchannel. DNA molecules were successfully driven by an electric field and separated to form bands in the range of 100 bp to 1 kbp in a 2.0% agarose-filled microchannel with 8 mm of effective separation length.     

Fabrication of lab-on chip platforms by hot embossing and photo patterning

In this paper, we review the approaches developed in our laboratory to fabricate polymer-based microfluidic devices to suit a range of applications in bio- or chemical analysis. Thermoplastic materials such as polycarbonate (PC) and poly(methyl methacrylate) (PMMA) are used to fabricate microfluidic devices via hot embossing. To emboss microchannels, we use hard stamps fabricated in silicon or soft stamps molded on poly(dimethylsiloxane) (PDMS). Hard stamps are fabricated on silicon wafers through photolithography and deep reactive ion etching (DRIE). Soft stamps are fabricated by casting PDMS prepolymer on silicon molds. To enclose the fluidic channels, direct fusion bonding was found to produce the highest bond strength with minimal structural deformation. One-step photolithographic methods have also been explored to produce via photochemical patterning microfluidic structures in photocurable materials. We use the photocurable capabilities of a PDMS copolymer, which incorporates a methacrylate crosslinker. Microfluidic channels are produced via one step-photopatterning processes by crosslinking the prepolymer mixture through a photomask. The smaller feature size attainable was 100 microm. Structures with higher spatial resolution are fabricated through a photoimprinting process whereby a mold is pressed against the precured mixture during UV crosslinking exposure. The application of the fabricated fluidic devices in electrophoretic ion analysis is also presented.     

Surface plasmon resonance imaging analysis of protein-protein interactions using on-chip-expressed capture protein

A surface plasmon resonance (SPR) imaging system, combined with a microwell gold chip for on-chip cell cultivation, was used to monitor protein-protein interactions. In particular, we developed an on-chip microscale cell cultivation system that integrates cell culture and on-chip analysis of protein-protein interactions on a single microwell chip in a time- and labor-saving manner. To assess the performance of this system in the analysis of protein-protein interactions, we conducted a series of protein-protein interaction analyses by measuring the binding of the yeast GAL4 dimerization domain (GAL4DD) to the GAL11 protein (GAL11P). Our system was found to enable the simple and rapid analysis of protein-protein interactions, requiring no special cell culturing equipment or recombinant protein expression prior to the immobilization of the purified proteins onto the chip. Our results demonstrate that the combination of an on-chip cell cultivation system and an SPR imaging system can be a useful tool to study protein-protein interactions without the need for time-consuming and labor-intensive protein preparation steps as well as fluorescent or other labeling of the interactants.     

Amine coupling versus biotin capture for the assessment of sulfonamide as ligands of hCA isoforms

This work was dedicated to the development of a reliable SPR method allowing the simultaneous and quick determination of the affinity and selectivity of designed sulfonamide derivatives for hCAIX and hCAXII versus hCAII, in order to provide an efficient tool to discover drugs for anticancer therapy of solid tumors. We performed for the first time a comparison of two immobilization approaches of hCA isoforms. First one relies on the use of an amine coupling strategy, using a CM7 chip to obtain higher immobilization levels than with a CM5 chip and consequently the affinity with an higher precision (CV% < 10%). The second corresponds to a capture of proteins on a streptavidin chip, named CAP chip, after optimization of biotinylation conditions (amine versus carboxyl coupling, biotin to protein ratio). Thanks to the amine coupling approach, only hCAII and hCAXII isoforms were efficiently biotinylated to reach relevant immobilization (3000 RU and 2700 RU, respectively) to perform affinity studies. For hCAIX, despite a successful biotinylation, capture on the CAP chip was a failure. Finally, concordance between affinities obtained for the three derivatives to CAs isozymes on both chips has allowed to valid the approaches for a further screening of new derivatives.     

Simultaneous detection of C-reactive protein and other cardiac markers in human plasma using micromosaic immunoassays and self-regulating microfluidic networks

We show a proof-of-concept in which we combine our previously published concepts of micromosaic immunoassays (microMIAs) with self-regulating microfluidic networks (microFNs) to detect C-reactive protein (CRP) and other cardiac markers such as myoglobin (Mb) and cardiac Troponin I (cTnI). The microFNs are microfabricated in Si, have a well-defined surface chemistry, and are affixed to a bibulous material so as to self-regulate the displacement of an aliquot of liquid through the microFNs using capillary forces. An open section of the channels of the microFNs is covered with a hydrophobic poly(dimethylsiloxane) (PDMS) slab that acts as the substrate for a solid-phase immunoassay. Here, individual assays are conducted using independent channels. These assays are "sequential": series of samples, reagents, and buffers are displaced one after the other over the PDMS surface, and, as these assays are conducted under "microfluidic" conditions, they are fast to perform, very economical in their use of reagents, extremely integrated, and yield high-quality signals. The combinatorial character of microMIAs is exploited to optimize the assay parameters for detecting CRP. In particular, we found it optimal to deposit the capture antibody for CRP on PDMS at a concentration between 20 and 500 microg ml(-1) in PBS in 1 min and to detect captured CRP in 2 min using a detection antibody having a concentration in PBS of 120 microg ml(-1). With this method, CRP is quantitatively detected within 10 min in one microliter of human plasma down to concentrations of 30 ng ml(-1), which suggests the possibility to detect CRP at clinically relevant concentrations for the management of coronary heart disease (CHD) and systemic inflammation.     

Phosphorylcholine Group-immobilized Surface Prepared on Polydimethylsiloxane Membrane by In Situ Reaction for Its Reduced Biofouling

To reduce interactions between biological molecules and the surface of microchip devices including the microchip, which should be conducted to improve sensitivity, reactivity, and the typical phospholipid polar group, the phosphorylcholine group-immobilized surfaces were prepared. The surface modification of polydimethylsiloxane (PDMS) was performed by in situ reaction during curing by cross-linking the PDMS prepolymers. Since it is known that 2-methacryloyloxyethyl phosphorylcholine (MPC) facilitates the preparation of biomedical polymers with excellent biocompatibility and antithrombogenicity, it was used as the reactant for surface modification. The MPC was coated on the glass substrate, and two-liquid-type PDMS prepolymers were then applied. During the curing process of the vinyl groups of poly(dimetylsiloxane-co-methylsiloxane) and poly(dimethylsiloxane-co-methylvinylsiloxane), the methacrylate group in MPC was attached onto the PDMS surface via a hydrosilyl group. Analysis of the surface characteristics by X-ray photoelectron spectroscopy and measurement of the surface contact angle revealed that the introduction of the phosphorylcholine group in the MPC unit on the surface induced hydrophilicity at the surface. Further, protein adsorption on the surface decreased with an increase in the number of phosphorylcholine groups. Based on these results, we concluded that the construction of the phosphorylcholine group-enriched surface on the PDMS substrate could be achieved by immobilization of MPC, and it may facilitate fabrication of biomedical devices, particularly microfluidic devices.     

Grating-coupled surface plasmon resonance: a cell and protein microarray platform

Grating-coupled surface plasmon resonance (GCSPR) is a method for the accurate assessment of analyte in a multiplexed format using small amounts of sample. In GCSPR, the analyte is flowed across specific receptors (e.g. antibodies or other proteins) that have been immobilized on a sensor chip. The chip surface is illuminated with p-polarized light that couples to the gold surface's electrons to form a surface plasmon. At a specific angle of incidence, the GCSPR angle, the maximum amount of coupling occurs, thus reducing the intensity of reflected light. Shifts in the GCSPR angle can be correlated with refractive index increases following analyte capture by chip-bound receptors. Because regions of the chip can be independently analyzed, this system can assess 400 interactions between analyte and receptor on a single chip. We have used this label-free system to assess a number of molecules of immunological interest. GCSPR can simultaneously detect an array of cytokines and other proteins using the same chip. Moreover, GCSPR is also compatible with assessments of antigen expression by intact cells, detecting cellular apoptosis and identifying T cells and B cells. This technology represents a powerful new approach to the analysis of cells and molecular constituents of biological samples.     

Complete amino acid sequence determinations demonstrate identity of the urinary Bence Jones protein (BJP-DIA) and the amyloid fibril protein (AL-DIA) in a case of AL-amyloidosis.

The complete primary structures of both the main amyloid fibril protein component (AL-DIA) and the soluble Bence Jones protein (BJP-DIA) obtained from the same patient with AL-amyloidosis are reported for the first time. The amino acid sequences were determined by automated Edman degradation following proteolytic digestion of the isolated proteins and HPLC separation of the resulting fragments and by amino-terminal sequencing after treatment with pyroglutamate aminopeptidase. Sequencing data were confirmed by amino acid analysis and plasma desorption mass spectrometry (PDMS). Molecular weights of the complete proteins were determined by laser desorption mass spectrometry. The amyloid fibril preparation contained a complete monoclonal lambda immunoglobulin light chain (subgroup 1.2) as well as different-sized fragments thereof which were identified by immunoblotting and amino-terminal sequencing following immobilization of electrophoretically-separated proteins on poly(vinylidene difluoride) (PVDF) membranes. The soluble urinary Bence Jones protein (BJP-DIA) was a dimer of monoclonal L-chains with a primary structure identical to that of the amyloid L-chain (AL-DIA) and thus represented the amyloid precursor protein.     

An approach for the improved immobilization of penicillin G acylase onto macroporous poly(glycidyl methacrylate‐co‐ethylene glycol dimethacrylate) as a potential industrial biocatalyst

The use of penicillin G acylase (PGA) covalently linked to insoluble carrier is expected to produce major advances in pharmaceutical processing industry and the enzyme stability enhancement is still a significant challenge. The objective of this study was to improve catalytic performance of the covalently immobilized PGA on a potential industrial carrier, macroporous poly(glycidyl methacrylate‐co‐ethylene glycol dimethacrylate) [poly(GMA‐co‐EGDMA)], by optimizing the copolymerization process and the enzyme attachment procedure. This synthetic copolymer could be a very promising alternative for the development of low‐cost, easy‐to‐prepare, and stable biocatalyst compared to expensive commercially available epoxy carriers such as Eupergit or Sepabeads. The PGA immobilized on poly(GMA‐co‐EGDMA) in the shape of microbeads obtained by suspension copolymerization appeared to have higher activity yield compared to copolymerization in a cast. Optimal conditions for the immobilization of PGA on poly(GMA‐co‐EGDMA) microbeads were 1 mg/mL of PGA in 0.75 mol/L phosphate buffer pH 6.0 at 25°C for 24 h, leading to the active biocatalyst with the specific activity of 252.7 U/g dry beads. Chemical amination of the immobilized PGA could contribute to the enhanced stability of the biocatalyst by inducing secondary interactions between the enzyme and the carrier, ensuring multipoint attachment. The best balance between the activity yield (51.5%), enzyme loading (25.6 mg/g), and stability (stabilization factor 22.2) was achieved for the partially modified PGA. © 2015 American Institute of Chemical Engineers Biotechnol. Prog., 32:43–53, 2016