Surface plasmon resonance (SPR) spectroscopy, a powerful tool for biosensing and protein interaction analysis, is currently confined to gold substrates and the relevant surface chemistries involving dextran and functional thiols. Drawbacks of using self-assembled monolayers (SAMs) for SPR-related surface modification include limited stability, pinhole defects, bioincompatibility, and nonspecific protein adsorption. Here we report the development of stable nanometer-scale glass (silicate) layers on gold substrates for SPR analysis of protein toxins. The nanoscale silicate layers were built up with layer-by-layer deposition of poly(allylamine hydrochloride) and sodium silicate, followed by calcination at high temperature. The resulting silicate films have a thickness ranging from 2 to 15 nm and demonstrate outstanding stability in flow cell conditions. The use of these surfaces as a platform to construct supported bilayer membranes (SBMs) is demonstrated, and improved performance against protein adsorption on SBM-coated surfaces is quantified by SPR measurements. SBMs can be formed reproducibly on the silicate surface via vesicle fusion and quantitatively removed using injection of 5% Triton X-100 solution, generating a fresh surface for each test. Membrane properties such as lateral diffusion of the SBMs on the silicate films are characterized with photobleaching methods. Studies of protein binding with biotin/avidin and ganglioside/cholera toxin systems show detection limits lower than 1 microg/mL (i.e., nanomolar range), and the response reproducibility is better than 7% RSD. The method reported here allows many assay techniques developed for glass surfaces to be transferred to label-free SPR analysis without the need for adaptation of protocols and time-consuming synthetic development of thiol-based materials and opens new avenues for developing novel bioanalytical technologies for protein analysis.

We demonstrate the characterization of mu-contact printed protein patterns and analysis of protein-protein interactions by two-dimensional (2-D) surface plasmon resonance imaging (SPRi). Advancements in SPRi image quality from employing a light emitting diode (LED) as the light source are described. We show that a LED offers an ideal point source that can eliminate interference artifacts and speckles found when using a laser source. The attainable thickness resolution in fixed-angle imaging is comparable to that of a monochromatic source, providing a solid foundation for quantitative analysis with the system. The SPR imaging technique reported here affords sub-nanometer thickness sensitivity and micrometer lateral resolution, allowing for convenient studies of biomolecular interactions and surface morphologies of ultrathin films. Spatially well-defined protein patterns of bacterial toxins were obtained by microcontact printing using a polydimethylsiloxane (PDMS) stamp on a functionalized self-assembled monolayer on Au. The influence of protein concentration in the inking solution on transfer efficiency was investigated, and a nonlinear correlation was observed between the solution concentration and the amount of protein immobilized on the surface. Quantitative analysis of protein interaction was performed with toxin-specific antibody, showing a concentration-dependent relationship that verifies the retention of biological activity of the protein after printing. The study demonstrates the feasibility and effectiveness of using LEDs as light sources in SPR imaging, opening doors for developing compact SPR instruments for direct, sensitive, and label-free detection of biohazardous molecules.

We report on the use of PDMS multichannels for affinity studies of DNA aptamer-human Immunoglobulin E (IgE) interactions by surface plasmon resonance imaging (SPRi). The sensing surface was prepared with thiol-terminated aptamers through a self-assembling process in the PDMS channels defined on a gold substrate. Cysteamine was codeposited with the thiol aptamers to promote proper spatial arrangement of the aptamers and thus maintain their optimal binding efficiencies. Four aptamers with different nucleic acid sequences were studied to test their interaction affinity toward IgE, and the results confirmed that aptamer I (5'-SH-GGG GCA CGT TTA TCC GTC CCT CCT AGT GGC GTG CCC C-3') has the strongest binding affinity. Control experiments were conducted with a PEG-functionalized surface and IgG was used to replace IgE in order to verify the selective binding of aptamer I to the IgE molecules. A linear concentration-dependent relationship between IgE and aptamer I was obtained, and a 2-nM detection limit was achieved. SPRi data were further analyzed by global fitting, and the dissociation constant of aptamer I-IgE complex was found to be 2.7 x 10(-7) M, which agrees relatively well with the values reported in the literature. Aptamer affinity screening by SPR imaging demonstrates marked advantages over competing methods because it does not require labeling, can be used in real-time, and is potentially high-throughput. The ability to provide both qualitative and quantitative results on a multichannel chip further establishes SPRi as a powerful tool for the study of biological interactions in a multiplexed format.

We report the fabrication and characterization of a micropatterned membrane electrode for electrochemical signaling of a bacterial pore-forming toxin, Streptolysin O (SLO) from S. pyogenes. Microcontact printing of an alkylthiol monolayer was used to fabricate an array template, onto which cholesterol-containing DMPC vesicles were fused to form lipid layer structures. The construction of the supported membranes, including pattern transfer and vesicle fusion, was characterized by in-situ surface plasmon resonance (SPR) imaging and electrochemistry. Quantitative analysis of the resulting membrane by using SPR angular shift measurements indicates that the membranes in the hydrophilic pockets have an average thickness of 8.2 +/- 0.4 nm. Together with fluorescence microscopy studies, the results suggest that this could be a mixed lipid assembly that may consist of a bilayer, vesicle fragments, and lipid junctions. The voltammetric response of the redox probe ferrocene carboxylic acid (FCA) was measured to quantify the toxin action on the supported membrane. The electrochemical measurements indicate that fusion of vesicles on the template blocked the access of FCA, whereas the injection of SLO toxin restored the redox response. The anodic peak current of FCA was found to increase with toxin concentration until a plateau was reached at 40 HU/mL. The method is highly sensitive such that 0.1 HU/mL of SLO (1.25 pM) can yield a well-defined response. In addition, it eliminates the need for a highly insulating layer in membrane sensing, which opens up new avenues in developing novel sensing interfaces for membrane-targeting proteins and peptides.

We report the multiplexed, simultaneous analysis of antigen-antibody interactions that involve human immunoglobulin G (IgG) on a gold substrate by the surface plasmon resonance imaging method. A multichannel, microfluidic chip was fabricated from poly(dimethylsiloxane) (PDMS) to selectively functionalize the surface and deliver the analyte solutions. The sensing interface was constructed using avidin as a linker layer between the surface-bound biotinylated bovine serum albumin and biotinylated anti-human IgG antibodies. Four mouse anti-human IgG antibodies were selected for evaluation and the screening was achieved by simultaneously monitoring protein-protein interactions under identical conditions. Antibody-antigen binding affinities towards human immunoglobulin were quantitatively compared by employing Langmuir adsorption isotherms for the analysis of SPRi responses obtained under equilibrium conditions. We were able to identify two IgG samples with higher affinities towards the target, and the determined binding kinetics falls within the typical range of values reported in the literature. Direct measurement of proteins in serum samples by SPR imaging was achieved by developing methods to minimize nonspecific adsorption onto the avidin-functionalized surface, and a limit of detection (LOD) of 6.7 nM IgG was obtained for the treated serum samples. The combination of SPR imaging and multichannel PDMS chips offers convenience and flexibility for sensitive and label-free measurement of protein-protein interactions in complex conditions and enables high-throughput screening of pharmaceutically significant molecules.

We report the development of an air-stable, supported membrane array by use of photolithography for label-free detection of lipid-protein interactions. Phosphoinositides and their phosphorylated derivatives (PIPs) were studied for their binding properties to proteins with lipid microarray in combination with surface plasmon resonance imaging (SPRi). We have demonstrated a simple method to fabricate lipid arrays using photoresist and carried out a series of surface characterizations with SPRi, ac impedance, cyclic voltammetry, and fluorescence microscopy to validate the array quality and lipid bilayer formation. A number of lipid compositions have been tested for the robustness of resulting membranes when undergoing dehydration and rehydration procedures, and the 1,2-dioleoyl-sn-glycero-3-ethylphosphocholine/poly(ethylene glycol)-phosphatidylethanolamine (DOPC+/PEG-PE) system stands out as the best performer that yields the recovery to within 2% of the original state according to SPR sensorgrams. Limits of detection on the dehydrated/rehydrated DOPC+/PEG-PE membranes were determined to be 33 nM for avidin binding to biotinylated lipids, 73.5 nM for cholera toxin to GM1, and 25 nM for PtdIns(4,5)P2-binding protein (P(4,5)BP) to PtdIns(4,5)P2 lipid, respectively. These results demonstrate the suitability and sensitivity of this membrane for constructing membrane arrays for SPRi analysis under ambient conditions. With the use of this addressable and functional lipid membrane array, the screening of specific lipid-protein interactions has been conducted. Strong and specific interactions between P(4,5)BP and PtdIns(4,5)P2/DOPC+/PEG-PE membrane were observed as expected, while cross reactions were spotted for P(4,5)BP/PtdIns(4)P and avidin/GM1 at varied degrees. The air-stable membrane array demonstrated here presents a simple, effective approach for constructing functional membrane surfaces for screening applications, which opens a new avenue for the label-free study of membrane proteins and other forms of lipid-membrane interactions.