Curved Membrane Mimics for Quantitative Probing of Protein-Membrane Interactions by Surface Plasmon Resonance.

A majority of biomimetic membranes used for current biophysical studies rely on planar structures such as supported lipid bilayer (SLB) and self-assembled monolayers (SAMs). While they have facilitated key information collection, the lack of curvature makes these models less effective for the investigation of curvature-dependent protein binding. Here, we report the development and characterization of curved membrane mimics on a solid substrate with tunable curvature and ease in incorporation of cellular membrane components for the study of protein-membrane interactions. The curved membranes were generated with an underlayer lipid membrane composed of DGS-Ni-NTA and POPC lipids on the substrate, followed by the attachment of histidine-tagged cholera toxin (his-CT) as a capture layer. Lipid vesicles containing different compositions of gangliosides, including GA1, GM1, GT1b, and GQ1b, were anchored to the capture layer, providing fixation of the curved membranes with intact structures. Characterization of the curved membrane was accomplished with surface plasmon resonance (SPR), fluorescence recovery after photobleaching (FRAP), and nano-tracking analysis (NTA). Further optimization of the interface was achieved through principal component analysis (PCA) to understand the effect of ganglioside type, percentage, and vesicle dimensions on their interactions with proteins. In addition, Monte Carlo simulations were employed to predict the distribution of the gangliosides and interaction patterns with single point and multipoint binding models. This work provides a reliable approach to generate robust, component-tuning, and curved membranes for investigating protein interactions more pertinently than what a traditional planar membrane offers.

Development of a surface plasmon resonance based immunosensor for diclofenac quantification in water.

A Surface Plasmon Resonance (SPR) biosensor based on an inhibition immunoassay was developed for the detection of diclofenac (DCF) in aqueous solution. Due to the small size of DCF, an hapten-protein conjugate was produced by coupling DCF to bovine serum albumin (BSA). DCF-BSA conjugate formation was confirmed via MALDI-TOF mass spectrometry. The resulting conjugate was immobilized onto the surface of a sensor fabricated via e-beam deposition of a 2 nm chromium adhesion layer followed by a 50 nm gold layer onto precleaned BK7 glass slides. Immobilization onto the nano thin gold surface was accomplished by covalent amide linkage through a self-assembled monolayer. Samples were composed of a mixture of antibody at a fixed concentration and DCF at different known concentrations in deionized water, causing the inhibition of anti-DCF on the sensor. The DCF-BSA was obtained with a ratio of 3 DCF molecules per BSA. A calibration curve was performed using concentrations between 2 and 32 µg L-1. The curve was fitted using the Boltzmann equation, reaching a limit of detection (LOD) of 3.15 µg L-1 and limit of quantification (LOQ) of 10.52 µg L-1, the inter-day precision was calculated and an RSD value of 1.96% was obtained; and analysis time of 10 min. The developed biosensor is a preliminary approach to the detection of DCF in environmental water samples, and the first SPR biosensor developed for DCF detection using a hapten-protein conjugate.

Detection of Multiple Sclerosis Biomarkers in Serum by Ganglioside Microarrays and Surface Plasmon Resonance Imaging.

Multiple sclerosis (MS) is an autoimmune disease that damages the myelin sheaths of nerve cells in the central nervous system. An individual suffering from MS produces increased levels of antibodies that target cell membrane components, such as phospholipids, gangliosides, and membrane proteins. Among them, anti-ganglioside antibodies are considered as important biomarkers to differentiate MS from other diseases that exhibit similar symptoms. We report here a label-free method for detecting a series of antibodies against gangliosides in serum by surface plasmon resonance imaging (SPRi) in combination with a carbohydrate microarray. The ganglioside array was fabricated with a plasmonically tuned, background-free biochip, and coated with a perfluorodecyltrichlorosilane (PFDTS) layer for antigen attachment as a self-assembled pseudo-myelin sheath. The chip was characterized with AFM and matrix-assisted laser desorption ionization mass spectrometry, demonstrating effective functionalization of the surface. SPRi measurements of patients' mimicking blood samples were conducted. A multiplexed detection of antibodies for anti-GT1b, anti-GM1, and anti-GA1 in serum was demonstrated, with a working range of 1 to 100 ng/mL, suggesting that it is well suited for clinical assessment of antibody abnormality in MS patients. Statistical analyses, including PLS-DA and PCA show the array allows comprehensive characterization of cross reactivity patterns between the MS specific antibodies and can generate a wide range of information compared to traditional end point assays. This work uses PFDTS surface functionalization and enables direct MS biomarker detection in serum, offering a powerful alternative for MS assessment and potentially improved patient care.

Plasmonic Biosensing with Aluminum Thin Films under the Kretschmann Configuration.

Aluminum has recently attracted considerable interest as a plasmonic material due to its unique optical properties, but most work has been limited to nanostructures. We report here SPR biosensing with aluminum thin-films using the standard Kretschmann configuration that has previously been dominated by gold films. Electron-beam physical vapor deposition (EBPVD)-prepared Al films oxidize in air to form a nanofilm of Al2O3, yielding robust stability for sensing applications in buffered solutions. FDTD simulations revealed a sharp plasmonic dip in the visible range that enables measurement of both angular shift and reflection intensity change at a fixed angle. Bulk and surface tests indicated that Al films exhibited superb sensitivity performance in both categories. Compared to Au, the Al/Al2O3 layer showed a marked effect of suppressing nonspecific binding from proteins in human serum. Further characterization indicated that Al film demonstrated a higher sensitivity and a wider working range than Au films when used for SPR imaging analysis. Combined with its economic and manufacturing benefits, the Al thin-film has the potential to become a highly advantageous plasmonic substrate to meet a wide range of biosensing needs in SPR configurations.

Expanding the scope of chemiluminescence in bioanalysis with functional nanomaterials.

Nanomaterial-enabled chemiluminescence (CL) detection has become a growing area of interest in recent years. We review the development of nanomaterial-based CL detection strategies and their applications in bioanalysis. Much progress has been achieved in the past decade, but most attempts still remain in the proof-of-concept stage. This review highlights recent advances in nanomaterials in CL detection and organizes them into three groups based on their role in detection: as a sensing platform, as a signal probe, and applications in homogeneous systems. Furthermore, we have discussed the critical challenges we are facing and future prospects of this field.

Multiplex immunoassay of chicken cytokines via highly-sensitive chemiluminescent imaging array.

Quantitative detection of multiple chicken cytokines is a good evaluation of cell-mediated immunity in chickens after disease infection or vaccination. However, current assay methods for chicken cytokines cannot meet the needs of clinical diagnosis due to unsatisfactory sensitivity and low assay throughput. Herein, a sensitive chemiluminescence (CL) imaging immunosensor array has been developed for high-throughput detection of multiple chicken cytokines. The chicken cytokines immunosensor array was prepared by assembling different cytokine capture antibodies onto a disposable silanized glass chip, where horseradish peroxidase and antibody-conjugated gold nanoparticles were used as multienzymatic amplification probe for CL imaging signal amplification. By using a sandwich assay mode, the amplified CL signals from each sensing array cell were collected for quantitation. Using chicken interleukin-4 and chicken interferon-γ as model cytokines, this novel multiplexed and amplified method demonstrated simultaneous measurement of the two chicken cytokines in the linear ranges of 0.008-0.12 ng/mL and 0.005-0.20 ng/mL, respectively, which yields limits of detection down to 2 pg/mL and 3 pg/mL. The CL imaging array method reported here also demonstrated high specificity, good repeatability, and high stability and accuracy, providing a novel multiplex immunoassay strategy for highly sensitive and high-throughput detection of chicken cytokines and further disease diagnosis in poultry.

Advances in Optical Sensing and Bioanalysis Enabled by 3D Printing.

The recent explosion of 3D printing applications in scientific literature has expanded the speed and effectiveness of analytical technological development. 3D printing allows for manufacture that is simply designed in software and printed in-house with nearly no constraints on geometry, and analytical methodologies can thus be prototyped and optimized with little difficulty. The versatility of methods and materials available allows the analytical chemist or biologist to fine-tune both the structural and functional portions of their apparatus. This flexibility has more recently been extended to optical-based bioanalysis, with higher resolution techniques and new printing materials opening the door for a wider variety of optical components, plasmonic surfaces, optical interfaces, and biomimetic systems that can be made in the laboratory. There have been discussions and reviews of various aspects of 3D printing technologies in analytical chemistry; this Review highlights recent literature and trends in their applications to optical sensing and bioanalysis.

Ultrasensitive Detection of Bacterial Protein Toxins on Patterned Microarray via Surface Plasmon Resonance Imaging with Signal Amplification by Conjugate Nanoparticle Clusters.

Sensitive detection and monitoring of biological interactions in a high throughput, multiplexed array format has numerous advantages. We report here a method to enhance detection sensitivity in surface plasmon resonance (SPR) spectroscopy and SPR imaging via the effect of accumulation of conjugated nanoparticles of varying sizes. Bacterial cholera toxin (CT) was chosen for the demonstration of enhanced immunoassay by SPR. After immobilization of CT on a gold surface, specific recognition is achieved by biotinylated anti-CT. The signal is amplified by the attachment of biotinylated 20 nm AuNP via streptavidin bridge, followed by attachment of 5 nm streptavidin-functionalized Fe3O4NP to the AuNP-biotin surface. The continuous surface binding of two differently sized conjugated nanoparticles effectively increases their packing density on surface and significantly improves SPR detection sensitivity, allowing quantitative measurement of CT at very low concentration. The dense packing of conjugated nanoparticles on the surface was confirmed by atomic force microscopy characterization. SPR imaging of the immunoassay for high-throughput analysis utilized an Au-well microarray that attenuated the background resonance interference on the resulting images. A calibration curve of conjugated nanoparticle binding signal amplification for CT detection based on surface coverage has been obtained that shows a correlation in a range from 6.31 × 10-16 to 2.51 × 10-13 mol/cm2 with the limit of detection of 5.01 × 10-16 mol/cm2. The absolute quantity of detection limit using SPR imaging was 0.25 fmol. The versatile nanoparticles and biotin-streptavidin interaction used here should allow adaptation of this enhancement method to many other systems that include DNA, RNA, peptides, and carbohydrates, opening new avenues for ultrasensitive analysis of biomolecules.

The role of leaf toughness on foraging efficiency in Angola black and white colobus monkeys (Colobus angolensis palliatus).

OBJECTIVES: Examining the relationships among foraging behavior, food mechanical properties, and masticatory morphology is a bourgeoning research topic among behavioral ecologists and functional morphologists. The purpose of this study was to assess the degree to which leaf toughness influences foraging efficiency with regard to ingestion rate, masticatory investment, and masticatory rate. METHODS: Diet and feeding data were collected on adults from three groups of Colobus angolensis palliatus in the Diani Forest, Kenya, from July 2014 to December 2015. Ingestion rates were estimated by counting the number of items consumed during feeding bouts and multiplying this value by the mean mass of a particular food item. The number of mastications was also counted during 3-5 minute focal periods. Mechanical toughness of commonly eaten young leaves (n = 27 species) and mature leaves (n = 13 species) was recorded using a toughness tester equipped with a razor blade. RESULTS: Ingestion rates (g/min) negatively correlated with leaf toughness (r(2) = 0.73; p < 0.01) while masticatory investment (chews/g) positively correlated with leaf toughness (r(2) = 0.72; p < 0.01). Chewing rate (chews/min) was remarkably consistent regardless of leaf species and toughness values (r(2) = 0.09; p = 0.07). DISCUSSION: Our findings highlight the degree to which toughness values can vary among leaves and how this variation can dramatically influence ingestion rates and chewing efficiency in black and white colobus monkeys. Studies that link food mechanical properties with oral processing behaviors will ultimately provide important context for understanding craniofacial and dentognathic traits in primates.

Limits to sustained energy intake. XXII. Reproductive performance of two selected mouse lines with different thermal conductance.

Maximal sustained energy intake (SusEI) appears limited, but the factors imposing the limit are disputed. We studied reproductive performance in two lines of mice selected for high and low food intake (MH and ML, respectively), and known to have large differences in thermal conductance (29% higher in the MH line at 21°C). When these mice raised their natural litters, their metabolisable energy intake significantly increased over the first 13 days of lactation and then reached a plateau. At peak lactation, MH mice assimilated on average 45.3% more energy than ML mice (222.9±7.1 and 153.4±12.5 kJ day(-1), N=49 and 24, respectively). Moreover, MH mice exported on average 62.3 kJ day(-1) more energy as milk than ML mice (118.9±5.3 and 56.6±5.4 kJ day(-1), N=subset of 32 and 21, respectively). The elevated milk production of MH mice enabled them to wean litters (65.2±2.1 g) that were on average 50.2% heavier than litters produced by ML mothers (43.4±3.0 g), and pups that were on average 27.2% heavier (9.9±0.2 and 7.8±0.2 g, respectively). Lactating mice in both lines had significantly longer and heavier guts compared with non-reproductive mice. However, inconsistent with the 'central limit hypothesis', the ML mice had significantly longer and heavier intestines than MH mice. An experiment where the mice raised litters of the opposing line demonstrated that lactation performance was not limited by the growth capacity of offspring. Our findings are consistent with the idea that the SusEI at peak lactation is constrained by the capacity of the mothers to dissipate body heat.

MK801-induced activated caspase-3 exhibits selective co-localization with GAD67.

Blockade of the N-methyl-d-aspartate receptor (NMDAR) in postnatal day 7 (P7) rats can promote rapid and robust induction of the pro-apoptotic marker activated caspase-3 (AC3) and loss of the GABAergic marker GAD67 at P56. Thus, we hypothesized that NMDAR blockade-induced AC3 occurs in GAD67 positive cells at P7. To test this idea, we injected P7 rat pups with vehicle or MK801 and after 8h (peak of AC3 induction) we examined brain sections for both AC3 and GAD67. Compared to vehicle, MK801 profoundly induced AC3 in all brain regions examined but co-expression of GAD67 in the same cells was not observed. However, in brain regions where punctate (synaptic) GAD67 was abundant (for example, layer IV of the somatosensory cortex), AC3 was robust. These data suggest that whereas somatic expression of AC3 and GAD67 may be non-overlapping, areas that exhibit punctate GAD67 (and are high in synaptic turnover) may be more vulnerable to MK801 exposure.