Fabrication of anionic sulfate-functionalized nanoparticles as an immunosensor by protein immobilization.

Anionic sulfate (SO(4)(-))-functionalized polystyrene (PS) nanoparticles were prepared by the thermal decomposition of potassium persulfate (KPS) in the presence of sodium tetraborate via emulsion polymerization. The presence of a SO(4)(-) group at a solid/liquid interface of a particle surface was confirmed by a zeta potential value of -40.6 mV as well as the shifting of S 2p spectra toward a lower-binding-energy region around 162.7 eV (2p(3/2)) and 164.4 eV (2p(1/2)) in X-ray photoelectron spectroscopy (XPS) analysis. The electrostatic attraction between positively charged antibodies of human immunoglobulin G (hIgG) and cardiac troponin I (cTnI) and negatively charged particle surfaces was accomplished. The atomic force microscopy (AFM) measurement and bicinchoninic acid (BCA) assay results show binding structure between hIgG and antibodies of hIgG (anti-hIgG) with a gradual increase in particle diameter to 152.6 nm (bare), 170.2 nm (hIgG), and 178.9 nm (hIgG/anti-hIgG). Surface coverage densities of 331.4 ng/cm(2) (hIgG) and 320.3 ng/cm(2) (cTnI) and the binding capacity of hIgG to HyLite-750-labeled Fab-specific anti-hIgG (approximately 81.2%) indicate that the majority of hIgG was immobilized with a Y-shaped orientation. The sandwich immunoassay results provide the evidence that the immunological activity of cTnI on the PS nanoparticle surface was retained because the binding activity of the cTnI-PS nanoparticle/cTnI (antigen)/detection cTnI-antibody reaction showed a 5-fold higher activity than that of the cTnI-PS nanoparticle/human serum albumin (HSA)/detection cTnI antibody used as a negative control.

Synthesis of anionic sulfate functionalized polystyrene latex and protein adsorption for application as a biosensor.

Bare and surface modified polystyrene nanoparticles with an anionic sulfate end group were synthesized by emulsion polymerization for adsorption of a protein (immunoglobulin G, IgG) by passive adsorption and electrostatic attraction for application as a biosensor. Dynamic light scattering (DLS) measurements confirmed the formation of an IgG-polystyrene latex complex, showing increased hydrodynamic diameter (D(h)) of approximately 178 nm (passive adsorption) and approximately 220 nm (electrostatic attraction) after IgG adsorption. An increased Zeta (zeta) potential value from -38.8 mV to -3.84 mV following IgG adsorption by electrostatic attraction also reflected the formation of the IgG-polystyrene latex complex. Solid phase immunodetection results demonstrated the formation of the IgG-polystyrene latex complex by showing fluorescent signals induced by FITC, which conjugated to IgG on the surface of the polystyrene. In addition, immunoassay results showed that adsorbed IgG on the polystyrene latex surface was not desorbed and maintained immunoreactivity after 1 month from the initial IgG-polystyrene latex complex formation.

Analysis of recombinant protein expression using localized surface plasmon resonance (LSPR).

The localized surface plasmon resonance (LSPR)-based optical biosensor using nano-structures of noble metals has been considered as a useful tool for label-free detection of DNA hybridization and protein-protein interactions. We fabricated LSPR-based optical biosensors using gold nano-islands (nominal thickness; 75 A) on glass substrates that were easily made using the conventional fabrication methods. The formation of gold nano-islands on glass substrates was realized by heat treatment of thin gold film deposited with a low deposition rate (approximately 0.05 A/s). The morphologies of sensor surfaces composed of gold nano-islands were observed using an atomic force microscope (AFM) with a non-contact mode. To investigate the sensing capacity of the gold nano-island sensor for the binding of proteins by affinity interactions, the streptavidin and biotin interaction was used as a model system. In addition, detection of recombinant glutathione-S-transferase (GST)-tagged human interleukin-6 (hIL6) expressed in Escherichia coli was carried out by LSPR. It is expected that the LSPR sensors composed of gold nano-islands can be an alternative to traditional methods such as SDS-polyacrylamide gel electrophoresis (SDS-PAGE) for fast analysis of protein expression.

Study of SU-8 to make a Ni master-mold: Adhesion, sidewall profile, and removal.

For disposable microfluidic devices, easy and inexpensive fabrication is essential. Consequently, replication of microfluidic devices, using injection molding or hot embossing, from a master-mold is widely used. However, the conventional master-mold fabrication technique is unsatisfactory in terms of time and costs. In this regard, direct Ni growth (electroplating) from a back plate is promising when the photoresist is well-defined. Here, we demonstrate the use of SU-8 as a photoresist to define the Ni-growth region. We accomplish this application by focusing on the adhesion, the sidewall profile, and the removal of SU-8: the adhesion is enhanced by controlling the exposure dose, the soft-baking time, and by choosing the adhesion-promoting layer; the sidewall profile is regulated by selecting the intensity of each exposed wavelength, showing an aspect ratio of up to 20.9; and, easy removal is achieved by choosing a proper photoresist-stripper. Using the master-mold fabricated by this method, we test the mechanical stability of the features according to the aspect ratio and length; in the hot embossing process, the features are stable in the aspect ratio of up to 5.8 at a length of 200 microm. In addition, the plastic devices fabricated from this method are applied to the passive stop valves, showing a capillary pressure (-0.2 to -7.2 kPa).

Wafer-scale fabrication of polymer-based microdevices via injection molding and photolithographic micropatterning protocols.

Because of their broad applications in biomedical analysis, integrated, polymer-based microdevices incorporating micropatterned metallic and insulating layers are significant in contemporary research. In this study, micropatterns for temperature sensing and microelectrode sets for electroanalysis have been implemented on an injection-molded thin polymer membrane by employing conventional semiconductor processing techniques (i.e., standard photolithographic methods). Cyclic olefin copolymer (COC) is chosen as the polymer substrate because of its high chemical and thermal stability. A COC 5-in. wafer (1-mm thickness) is manufactured using an injection molding method, in which polymer membranes (approximately 130 microm thick and 3 mm x 6 mm in area) are implemented simultaneously in order to reduce local thermal mass around micropatterned heaters and temperature sensors. The highly polished surface (approximately 4 nm within 40 microm x 40 microm area) of the fabricated COC wafer as well as its good resistance to typical process chemicals makes it possible to use the standard photolithographic and etching protocols on the COC wafer. Gold micropatterns with a minimum 5-microm line width are fabricated for making microheaters, temperature sensors, and microelectrodes. An insulating layer of aluminum oxide (Al2O3) is prepared at a COC-endurable low temperature (approximately 120 degrees C) by using atomic layer deposition and micropatterning for the electrode contacts. The fabricated microdevice for heating and temperature sensing shows improved performance of thermal isolation, and microelectrodes display good electrochemical performances for electrochemical sensors. Thus, this novel 5-in. wafer-level microfabrication method is a simple and cost-effective protocol to prepare polymer substrate and demonstrates good potential for application to highly integrated and miniaturized biomedical devices.

Holographic polarization-selective module based on a small dove prism coupler for magneto-optical pickup heads.

A new structure for polarization-selective elements, consisting of two holographic gratings and a Dove prism coupler, is proposed. The absence of a multistage waveguide and the benefits of compact size and lightweight volume are the outstanding features of the new structure. Based on the coupled-wave theory, the analysis and design of the structure are discussed in detail to calculate the required index modulation. Several parameters, such as the recording intensity, the exposure time, and the recording angles for the fabrication of the proposed element, are determined. Under these conditions, the element is fabricated in Dupont photopolymer HRF-150-38 material and with an operating wavelength of 532 nm. A simplified pickup head is constructed to evaluate the performance of the fabricated element.

Multichannel surface plasmon resonance imaging and analysis of micropatterned self-assembled monolayers and protein affinity interactions.

Multichannel images of 11-mercaptoundecanoic acid and 11-mercapto-1-undecanol self-assembled monolayers together with a biospecific interferon-gamma (IFN-gamma)/anti-IFN-gamma antibody immunoaffinity interaction were observed by the two-dimensional surface plasmon resonance (2D-SPR) imaging system. With the fabricated 2D-SPR imaging system, adopting a white light source in combination with a narrow band-pass filter, sharp images were resolved, minimizing the diffraction patterns on the resulting images. Micropatterning of self-assembled monolayers was acheived by exploiting the UV photolysis of thiol bonding, instead of conventional photolithography. The line profile calibration of the image contrast with ellipsometric analysis enabled us to discriminate the change in monolayer thickness within a sub-nanometer scale. For the protein interactions on the surface, the biospecific affinity recognition reaction of IFN-gamma antigen with surface-immobilized antibody was analyzed. Through the signal amplification strategy based on the enzyme-catalyzed precipitation reaction in a sandwich-type immunoassay, biospecific antigen binding was found detectable down to a concentration of 1 ng/mL.

A polymer-based microfluidic device for immunosensing biochips.

This paper describes the design, fabrication, and test of a PDMS/PMMA-laminated microfluidic device for an immunosensing biochip. A poly(dimethyl siloxane)(PDMS) top substrate molded by polymer casting and a poly(methyl methacrylate)(PMMA) bottom substrate fabricated by hot embossing are bonded with pressure and hermetically sealed. Two inlet ports and an air vent are opened through the PDMS top substrate, while gold electrodes for electrochemical biosensing are patterned onto the PMMA bottom substrate. The analyte sample is loaded from the sample inlet port to the detection chamber by capillary force, without any external intervening forces. For this and to control the time duration of sample fluid in each compartment of the device, including the inlet port, diffusion barrier, reaction chamber, flow-delay neck, and detection chamber, the fluid conduit has been designed with various geometries of channel width, depth, and shape. Especially, the fluid path has been designed so that the sample flow naturally stops after filling the detection chamber to allow sufficient time for biochemical reaction and subsequent washing steps. As model immunosensing tests for the microfluidic device, functionalizations of ferritin and biotin to the sensing surfaces on gold electrodes and their biospecific interactions with antiferritin antiserum and streptavidin have been investigated. An electrochemical detection method for immunosensing by biocatalyzed precipitation has been developed and applied for signal registration. With the biochip, the whole immunosensing processes could be completed within 30 min.