Journal of Laboratory Automation

Microarrays simultaneously screen tens to thousands of biosamples to observe biochemical activities in protein—protein, protein—nucleic acid and small molecule interactions. In this high throughput analysis, rapid and reliable printing technologies are highly desired with less deterioration on biosamples during process. This study introduces several micro-contact printing systems to print out multiple proteins simultaneously, uniformly and continuously with batch-filling capability for rapid microarray formation, with very gentle process for biosample preservation. This printing system consists of two chips, including a micro-filling chip and a micro-stamp chip, for rapid/accurate registration and batch operation. The micro-filling chip can simultaneously transfer numerous protein solutions into the micro-stamp chip in seconds by capillary force without cross-contamination, while preserving the functionality of proteins. Different proteins can be dispensed into the corresponding channels and driven into the tips of the micro stamps. The micro stamp can be then brought to contact with the substrate to produce bio-fluid spot arrays. These devices have a potential to be expanded to a high throughput system for simultaneously printing hundreds of bio-fluid spots for hundreds times in minutes, and to form dense bio-microarrays for disease diagnosis or drug screening.

Nanoscale copolymer membranes that mimic the innate structure and properties of biological lipid membranes possessing hydrophilic and hydrophobic elements to support protein folding were used for a fundamental examination of protein—polymer integration. This study has integrated the neural synaptotagmin II (Syt II) protein, a documented target of the hemagglutinin-33 (Hn-33) protein associated with botulinum neurotoxin type A during the infection process, into polymethyloxazoline—polydimethylsiloxane—polymethyloxazoline nanomembranes. By integrating Syt II into block copolymer membranes, we have developed a neural mimetic membrane toward Hn-33 targeting the applications in nanomaterial-mediated detection. This technology can serve as a robust stand-alone platform for toxin diagnostic studies, or as a coating for integration with micro-/nanofabricated devices and electrodes for protein—protein interaction-based detection. To assess enhanced membrane complexity and toxin specificity, studies assessing the co-insertion of trisialoganglioside-GT1b (GT1b) and Syt II into the nanomembranes were used as a subsequent platform for botulinum neurotoxin type B detection. Protein—membrane integration was confirmed with atomic force microscopy imaging, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and Langmuir isotherm analysis.

Amphiphilic block copolymers are finding increased potential in biological and medical research due to their innate alternating hydrophilic and hydrophobic blocks/segments that can be used to package therapeutics, or coat a broad array of biological interfaces. Some studies are already directed toward using these copolymers' ability to form micelles or vesicles to develop novel methods of drug delivery to prevent inflammation or pro-cancer activity. Our study, however, aims to investigate the more fundamental cell—block copolymer interaction for use in protective nanofilms to prevent biofouling of non-tissue-based implantable devices. Block copolymers could potentially fill the demand for biologically inert, highly functionalizable biomaterials desirable for this type of application. Two such polymers used in our study include polymethyloxazoline—polydimethylsiloxane—polymethyloxazoline (PMOXA—PDMS—PMOXA) triblock copolymer and polyethylene oxide-poly(methyl methacrylate) (PEO—PMMA) diblock copolymer. Each block copolymer possesses hydrophilic and hydrophobic blocks that enable it to mimic the cell lipid membrane. So far we have shown that triblock copolymer is capable of inhibiting the accumulation of murine macrophages onto glass substrates. Preliminary evidence has suggested that the triblock copolymer has anti-adsorptive and noninflammatory capabilities during short incubation periods (7 days) in vitro. While the diblock copolymer displays minimal anti-adsorptive activities, nanofilms comprising a mixture of the two copolymers were able to significantly reduce macrophage accumulation onto glass substrates. The disparate behavior of macrophages on the different materials may be due to specific inherent properties such as preference for hydrophobic versus hydrophilic surfaces and/or rough versus smooth nanotextures. Furthermore, the specific endgroups of the two polymers may exhibit varying capacities to resisting non-specific protein adsorption. Continued investigation outlining the physical and chemical properties desirable for an anti-adsorptive nanofilm coating will serve as a basis to design durable implant—tissue interfaces that can react to various external stimuli.

We developed cost-effective and high-throughput techniques to fabricate metal nanostructure arrays of various geometries on solid substrates. Surface plasmons of these nanostructure arrays were investigated both experimentally and theoretically. We systematically studied the effects of different parameters on the localized surface plasmon resonance of the nanostructure arrays. We further developed a few approaches to tailor surface plasmons for different applications. As an example of the applications of these nanostructure arrays, we demonstrated all-optical plasmonic switches/modulators based on long-range ordered Au nanodisk arrays and photoresponsive liquid crystals. The advantages of such arrays include low-cost, high-throughput, and tailorable plasmonic properties. These arrays can serve as a platform that will stimulate further progress in both fundamental research and engineering applications of plasmonics.

Anovel in-plane microfluidic mixer based on fluidic discretization using vortex micropumps integrated in an optically transparent microfluidic substrate is presented in this article. The design, fabrication, simulation, and experimental results are described of this integrated micromixer. The basic working principle of the discretized fluidic mixer is to manipulate fluids as discretized volumes and inject them to an expansion chamber. Due to increase interfacial surface area of the discretized fluid “chunks,” the diffusion between these fluids can be completed in a shorter time, and the fluids can be mixed instantly without additional external energy. A numerical simulation was performed to emulate the flow field and mixing phenomenon to understand the results obtained by various flow experiments. Experimental results of discretized mixing have been successfully shown to have almost an ideal mixing performance and shown reasonably good match with the simulation results. Moreover, a dimensionless governing parameter (mixing index) was used to estimate the mixing performance in our system. This parameter is shown to be useful for the design and analyses of discretized mixing systems. Because this discretized mixing system requires simple mechanical structures, it provides flexibility for integrate with other microfluidic components. Also, optically transparent and biocompatible material was used to fabricate the microfluidic system, hence this micromixing system could be used to develop future fully automated biomedical and chemical “lab-on-chip” systems.

In this article, two-dimensional hexamethyldisilazane (HMDS) micropatterns were generated on glass substrates using photolithographic techniques for the assembly of functional proteins. The non-HMDS patterned areas were backfilled with poly(ethylene glycol) (PEG) silane to reduce the nonspecific protein adsorption. The hydrophobic methyl-terminated HMDS monolayer was verified to be favorable for physical protein adsorption with bovine serum albumin (BSA). The PEG-silane derivatized surface significantly reduced the BSA nonspecific binding by 97% compared to the pristine glass substrate so that high patterning selectivity was achieved. A universal streptavidin template was generated using preadsorbed biotinylated BSA on HMDS surface to sequentially bind additional biotinylated antibodies. Using this patterning strategy, the biotinylated goat anti-mouse (biotin-GAM) antibodies can be specifically recognized by the fluorescently labeled mouse immunoglobulin G, which indicated that the immobilized biotin-GAM was still bioactive. Also, the immobilized alkaline phosphatase was demonstrated to retain its enzymatic functionality by the ability to convert its fluorogenic substrate fluorescein diphosphate into fluorescent products. This simple and effective protein patterning technique can also be extended to create nanoscale protein arrays. Additionally, its adaptability for the assembly of arbitrary proteins and antibodies provides great potentials for biosensor and biomicroelectromechanical systems (MEMS) applications.

Emerging molecular studies have shown that the transcription factor NF-E2-related factor (Nrf2) plays an essential role in cancer chemoprevention. Here, we report the development of a molecular biosensor for rapid detection of antioxidant-responsive element (ARE)-bound Nrf2 protein. The development will provide a molecular assay for high-throughput screening of chemopreventive compounds. Specifically, a double-stranded DNA probe is designed based on the ARE sequence. One of the DNA strands is labeled with a fluorophore on the 5′ end and the complementary strand is labeled with a quencher on the 3′ end. A single-stranded DNA competitor is also designed. The existence of the Nrf2 stabilizes the fluorescent probes and delays the competitor from separating the fluorophore-quencher complex. Therefore, the concentration of the Nrf2 proteins can be measured quantitatively based on the fluorescence intensity. The molecular binding scheme was demonstrated using purified p50 and the detection of endogenous Nrf2 was demonstrated using whole-cell lysates treated with sulforaphane. The assay can easily be incorporated into an automated platform for high-throughput screening of chemopreventive compounds targeting Nrf2.