Enhanced electrochemical biosensing on gold electrodes with a ferri/ferrocyanide redox couple.

Detection of specific DNA is important in many fields. Label-free DNA sensing performed by electrochemical impedance spectroscopy (EIS) or using a quartz crystal microbalance (QCM) is widely employed for this purpose. Gold electrodes are mainly used for these techniques due to their chemical stability. However, ferro/ferricyanide used as a redox couple was found to etch the gold electrode and this significantly limited the repeatability of the EIS measurement. Inductively coupled plasma mass spectrometry (ICP-MS) and QCM experiments provided important clues about the gold dissolution mechanism and revealed that phosphate buffer promotes the dissolution of gold in the presence of the ferri/ferrocyanide redox couple. Tris buffered conditions, which provide the most stable environment, enabled the investigation of experimental parameters with a Q-sense electrochemistry module (QEM), which can perform QCM and EIS measurements simultaneously and revealed the principal factors that influence changes in the impedance. With the reproducible measurements, the estimation of an optimum probe-DNA concentration for detecting complementary DNA is demonstrated. In order to amplify the detection signal of target DNA, we sought to maximize the difference in response between the probe-only and target DNA by controlling the concentration of probe DNA. We showed that an intermediate probe-DNA concentration yields optimum signal amplification.

Polymer sieving matrices in microanalytical electrophoresis.

Microfluidic design has advanced existing protein separation capabilities and supported novel assays. Key metrics for successful protein separations include fast, robust, and sensitive analysis of complex mixtures of bio-macromolecules. Attaining high separation resolution is a chief concern. Here we review recent advances in polymer-based electrophoresis sieving materials that are impacting microfluidic bioanalytical applications. Looking forward, we comment on unmet needs for advanced separation media in micro-to-nanoscale devices.

Microchamber Western blotting using poly-L-lysine conjugated polyacrylamide gel for blotting of sodium dodecyl sulfate coated proteins.

We report a novel strategy to immobilize sodium dodecyl sulfate (SDS)-coated proteins for fully integrated microfluidic Western blotting. Polyacrylamide gel copolymerized with a cationic polymer, poly-L-lysine, effectively immobilizes all sized proteins after sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), and enables SDS-PAGE and subsequent immuno-probing in an automated microfluidic chip. Design of a poly-l-lysine conjugated polyacrylamide gel allows optimization of SDS-protein immobilization strength in the blotting gel region of the microchamber. The dependence of protein capture behavior on both the concentration of copolymerized charges and poly-lysine length is studied and gives important insight into an electrostatic immobilization mechanism. Based on analysis of protein conformation, the immobilized proteins bind with partner antibody after SDS dilution. We demonstrate each step of the microchamber Western blot, including injection, separation, transfer, immobilization, blocking, and immunoblot. The approach advances microfluidic protein immunoblotting, which is directly relevant to the widely-used SDS-PAGE based slab-gel Western blot, while saving sample volume, labor, and assay time.

DNA-Tethered Lipid Membrane Formation via Solvent-Assisted Self-Assembly.

DNA-tethered lipid bilayers have been used in many studies, based on the controllable and well-defined properties of DNA tethers. However, their application has been limited, because it is difficult to cover a wide range of surfaces and achieve electrical insulation. We implemented an existing method, where a DNA hybrid chip on a silica or glass surface supports a lipid membrane using solvent-assisted self-assembly. The formation of a continuous lipid bilayer was confirmed through the change in quartz crystal microbalance dissipation results, depending on the presence or absence of DNA hybrids. The fluidity of the DNA-tethered lipid membranes was analyzed using a fluorescence microscope. The electrochemical analysis demonstrated the versatility of this new technique, which can be used for sensor or electrode surface modification for biosensors or bioelectronics.

Vesicle fusion observed by content transfer across a tethered lipid bilayer.

Synaptic transmission is achieved by exocytosis of small, synaptic vesicles containing neurotransmitters across the plasma membrane. Here, we use a DNA-tethered freestanding bilayer as a target architecture that allows observation of content transfer of individual vesicles across the tethered planar bilayer. Tethering and fusion are mediated by hybridization of complementary DNA-lipid conjugates inserted into the two membranes, and content transfer is monitored by the dequenching of an aqueous content dye. By analyzing the diffusion profile of the aqueous dye after vesicle fusion, we are able to distinguish content transfer across the tethered bilayer patch from vesicle leakage above the patch.

Stability of DNA-tethered lipid membranes with mobile tethers.

We recently introduced two approaches for tethering planar lipid bilayers as membrane patches to either a supported lipid bilayer or DNA-functionalized surface using DNA hybridization (Chung, M.; Lowe, R. D.; Chan, Y-H. M.; Ganesan, P. V.; Boxer, S. G. J. Struct. Biol.2009, 168, 190-9). When mobile DNA tethers are used, the tethered bilayer patches become unstable, while they are stable if the tethers are fixed on the surface. Because the mobile tethers between a patch and a supported lipid bilayer offer a particularly interesting architecture for studying the dynamics of membrane-membrane interactions, we have investigated the sources of instability, focusing on membrane composition. The most stable patches were made with a mixture of saturated lipids and cholesterol, suggesting an important role for membrane stiffness. Other factors such as the effect of tether length, lateral mobility, and patch membrane edge were also investigated. On the basis of these results, a model for the mechanism of patch destruction is developed.

Capacitively coupled contactless conductivity detection for microfluidic capillary isoelectric focusing.

We report capacitively coupled contactless conductivity detection (C4D) of proteins separated by microfluidic capillary isoelectric focusing (µCIEF). To elucidate the evolution of negative conductivity peaks during focusing and seek IEF conditions for sensitive conductivity detection, numerical simulation was performed using a model protein GFP (green fluorescence protein) and hypothetical carrier ampholytes (CAs). C4D was successfully applied to the µCIEF by optimizing assay conditions using a simple and effective pressure-mobilization approach. The conductivity and fluorescence signals of a focused GFP band were co-detected, confirming that the obtained negative C4D peak could be attributed to the actual protein, not the non-uniform background conductivity profile of the focused CAs. GFP concentrations of 10 nM-30 µM was quantified with a detection limit of 10 nM. Finally, the resolving power was analyzed by separating a mixture of R-phycoerythrin (pI 5.01), GFP-F64L (pI 5.48), and RK-GFP (pI 6.02). The conductivities of the three separated fluorescence proteins were measured with average separation resolution of 2.06. We expect the newly developed label-free µCIEF-C4D technique to be widely adopted as a portable, electronics-only protein-analysis tool.

DNA-tethered membranes formed by giant vesicle rupture.

We have developed a strategy for preparing tethered lipid bilayer membrane patches on solid surfaces by DNA hybridization. In this way, the tethered membrane patch is held at a controllable distance from the surface by varying the length of the DNA used. Two basic strategies are described. In the first, single-stranded DNA strands are immobilized by click chemistry to a silica surface, whose remaining surface is passivated to prevent direct assembly of a solid supported bilayer. Then giant unilamellar vesicles (GUVs) displaying the antisense strand, using a DNA-lipid conjugate developed in earlier work [Chan, Y.-H.M., van Lengerich, B., et al., 2008. Lipid-anchored DNA mediates vesicle fusion as observed by lipid and content mixing. Biointerphases 3 (2), FA17-FA21], are allowed to tether, spread and rupture to form tethered bilayer patches. In the second, a supported lipid bilayer displaying DNA using the DNA-lipid conjugate is first assembled on the surface. Then GUVs displaying the antisense strand are allowed to tether, spread and rupture to form tethered bilayer patches. The essential difference between these methods is that the tethering hybrid DNA is immobile in the first, while it is mobile in the second. Both strategies are successful; however, with mobile DNA hybrids as tethers, the patches are unstable, while in the first strategy stable patches can be formed. In the case of mobile tethers, if different length DNA hybrids are present, lateral segregation by length occurs and can be visualized by fluorescence interference contrast microscopy making this an interesting model for interactions that occur in cell junctions. In both cases, lipid mobility is high and there is a negligible immobile fraction. Thus, these architectures offer a flexible platform for the assembly of lipid bilayers at a well-defined distance from a solid support.

Influence of natural organic matter (NOM) coatings on nanoparticle adsorption onto supported lipid bilayers.

As the worldwide usage of nanoparticles in commercial products continues to increase, there is growing concern about the environmental risks that nanoparticles pose to biological systems, including potential damage to cellular membranes. A detailed understanding of how different types of nanoparticles behave in environmentally relevant conditions is imperative for predicting and mitigating potential membrane-associated toxicities. Herein, we investigated the adsorption of two popular nanoparticles (silver and buckminsterfullerene) onto biomimetic supported lipid bilayers of varying membrane charge (positive and negative). The quartz crystal microbalance-dissipation (QCM-D) measurement technique was employed to track the adsorption kinetics. Particular attention was focused on understanding how natural organic matter (NOM) coatings affect nanoparticle-bilayer interactions. Both types of nanoparticles preferentially adsorbed onto the positively charged bilayers, although NOM coatings on the nanoparticle and lipid bilayer surfaces could either inhibit or promote adsorption in certain electrolyte conditions. While past findings showed that NOM coatings inhibit membrane adhesion, our findings demonstrate that the effects of NOM coatings are more nuanced depending on the type of nanoparticle and electrolyte condition. Taken together, the results demonstrate that NOM coatings can modulate the lipid membrane interactions of various nanoparticles, suggesting a possible way to improve the environmental safety of nanoparticles.

Note: A microfluidic freezer based on evaporative cooling of atomized aqueous microdroplets.

We report for the first time water-based evaporative cooling integrated into a microfluidic chip for temperature control and freezing of biological solution. We opt for water as a nontoxic, effective refrigerant. Aqueous solutions are atomized in our device and evaporation of microdroplets under vacuum removes heat effectively. We achieve rapid cooling (-5.1 °C/s) and a low freezing temperature (-14.1 °C). Using this approach, we demonstrate freezing of deionized water and protein solution. Our simple, yet effective cooling device may improve many microfluidic applications currently relying on external power-hungry instruments for cooling and freezing.

A Simple and Fast Aqueous-Phase Synthesis of Ultra-Highly Concentrated Silver Nanoparticles and Their Catalytic Properties.

A simple and fast synthetic route to ultra-highly concentrated silver nanoparticles with long-term stability by reducing AgNO3 with ascorbic acid in the presence of polyethyleneimine (PEI) as a stabilizer in an aqueous phase is reported. The concentration of silver precursor was as high as 2000 mm (200 g of Ag nanoparticle per liter of water) and the reaction time was less than 10 min. The resulting silver nanoparticles show long-term stability after two months of storage at room temperature without any signs of particle aggregation or precipitation in an aqueous phase. The successful ligand exchange of PEI-stabilized silver nanoparticles to polyethylene glycol (PEG) and polyvinylpyrrolidone (PVP) without particle aggregation is also demonstrated. In addition, the catalytic activities of silver nanoparticles stabilized by various stabilizers prepared by the ligand exchange method was investigated. The PEI-stabilized silver nanoparticles exhibited a higher stability than those of PEG- and PVP-stabilized silver nanoparticles in the diffusion-controlled catalytic reduction of 4-nitrophenol to 4-aminophenol by NaBH4 .

Formation and analysis of topographical domains between lipid membranes tethered by DNA hybrids of different lengths.

We recently described a strategy to prepare DNA-tethered lipid membranes either to fixed DNA on a surface or to DNA displayed on a supported bilayer [Boxer et al., J. Struct. Biol., 2009, 168, 190; Boxer et al., Langmuir, 2011, 27, 5492]. With the latter system, the DNA hybrids are laterally mobile; when orthogonal sense-antisense pairs of different lengths are used, the DNA hybrids segregate by height and the tethered membrane deforms to accommodate the height difference. This architecture is particularly useful for modelling interactions between membranes mediated by molecular recognition and resembles cell-to-cell junctions. The length, affinity and population of the DNA hybrids between the membranes are completely controllable. Interesting patterns of height segregation are observed by fluorescence interference contrast microscopy. Diverse behavior is observed in the segregation and pattern forming process and possible mechanisms are discussed. This model system captures some of the essential physics of synapse formation and is a step towards understanding lipid membrane behaviour in cell-to-cell junctions.

Use of monoclonal antibodies that recognize p60 for identification of Listeria monocytogenes.

Listeria monocytogenes causes major food-borne outbreaks of disease worldwide. Specific identification of this microorganism is of utmost importance to public health and industry. Listeria species are known to secrete a 60-kDa protein collectively termed p60, which is encoded by the iap (invasion-associated protein) gene and secreted in large quantities into the growth media. p60 is a highly immunogenic murein hydrolase that is essential for cell division. Due to these properties, p60 is an ideal diagnostic target for the development of immunological detection systems for L. monocytogenes. We report here two independent lines of monoclonal antibody (MAb): p6007, which specifically recognizes L. monocytogenes p60, and p6017, which reacts with a wide range of Listeria p60 proteins. By combining these antibodies with a polyclonal antibody, we developed efficient sandwich enzyme-linked immunosorbent assay (ELISA) systems which can specifically identify L. monocytogenes or generally detect Listeria species. Since an excess amount of the peptide corresponding to PepA or PepD did not interfere with the ELISA, and direct ELISAs were unable to detect both peptides, we concluded that the epitope presumed to be recognized by p6007 or p6017 could be distinguished from PepA and PepD as described by Bubert et al. (Appl. Environ. Microbiol. 60:3120-3127, 1997). To our best knowledge, this is the first example of an immunological identification system that uses p60-recognizing MAbs.