Single shot detection of alterations across multiple ionic currents from assimilation of cell membrane dynamics.

The dysfunction of ion channels is a causative factor in a variety of neurological diseases, thereby defining the implicated channels as key drug targets. The detection of functional changes in multiple specific ionic currents currently presents a challenge, particularly when the neurological causes are either a priori unknown, or are unexpected. Traditional patch clamp electrophysiology is a powerful tool in this regard but is low throughput. Here, we introduce a single-shot method for detecting alterations amongst a range of ion channel types from subtle changes in membrane voltage in response to a short chaotically driven current clamp protocol. We used data assimilation to estimate the parameters of individual ion channels and from these we reconstructed ionic currents which exhibit significantly lower error than the parameter estimates. Such reconstructed currents thereby become sensitive predictors of functional alterations in biological ion channels. The technique correctly predicted which ionic current was altered, and by approximately how much, following pharmacological blockade of BK, SK, A-type K+ and HCN channels in hippocampal CA1 neurons. We anticipate this assay technique could aid in the detection of functional changes in specific ionic currents during drug screening, as well as in research targeting ion channel dysfunction.

Noise-activated barrier crossing in multiattractor dissipative neural networks.

Noise-activated transitions between coexisting attractors are investigated in a chaotic spiking network. At low noise level, attractor hopping consists of discrete bifurcation events that conserve the memory of initial conditions. When the escape probability becomes comparable to the intrabasin hopping probability, the lifetime of attractors is given by a detailed balance where the less coherent attractors act as a sink for the more coherent ones. In this regime, the escape probability follows an activation law allowing us to assign pseudoactivation energies to limit cycle attractors. These pseudoenergies introduce a useful metric for evaluating the resilience of biological rhythms to perturbations.

Estimation of neuron parameters from imperfect observations.

The estimation of parameters controlling the electrical properties of biological neurons is essential to determine their complement of ion channels and to understand the function of biological circuits. By synchronizing conductance models to time series observations of the membrane voltage, one may construct models capable of predicting neuronal dynamics. However, identifying the actual set of parameters of biological ion channels remains a formidable theoretical challenge. Here, we present a regularization method that improves convergence towards this optimal solution when data are noisy and the model is unknown. Our method relies on the existence of an offset in parameter space arising from the interplay between model nonlinearity and experimental error. By tuning this offset, we induce saddle-node bifurcations from sub-optimal to optimal solutions. This regularization method increases the probability of finding the optimal set of parameters from 67% to 94.3%. We also reduce parameter correlations by implementing adaptive sampling and stimulation protocols compatible with parameter identifiability requirements. Our results show that the optimal model parameters may be inferred from imperfect observations provided the conditions of observability and identifiability are fulfilled.

Optimal solid state neurons.

Bioelectronic medicine is driving the need for neuromorphic microcircuits that integrate raw nervous stimuli and respond identically to biological neurons. However, designing such circuits remains a challenge. Here we estimate the parameters of highly nonlinear conductance models and derive the ab initio equations of intracellular currents and membrane voltages embodied in analog solid-state electronics. By configuring individual ion channels of solid-state neurons with parameters estimated from large-scale assimilation of electrophysiological recordings, we successfully transfer the complete dynamics of hippocampal and respiratory neurons in silico. The solid-state neurons are found to respond nearly identically to biological neurons under stimulation by a wide range of current injection protocols. The optimization of nonlinear models demonstrates a powerful method for programming analog electronic circuits. This approach offers a route for repairing diseased biocircuits and emulating their function with biomedical implants that can adapt to biofeedback.

Dual Mechanism for the Emergence of Synchronization in Inhibitory Neural Networks.

During cognitive tasks cortical microcircuits synchronize to bind stimuli into unified perception. The emergence of coherent rhythmic activity is thought to be inhibition-driven and stimulation-dependent. However, the exact mechanisms of synchronization remain unknown. Recent optogenetic experiments have identified two neuron sub-types as the likely inhibitory vectors of synchronization. Here, we show that local networks mimicking the soma-targeting properties observed in fast-spiking interneurons and the dendrite-projecting properties observed in somatostatin interneurons synchronize through different mechanisms which may provide adaptive advantages by combining flexibility and robustness. We probed the synchronization phase diagrams of small all-to-all inhibitory networks in-silico as a function of inhibition delay, neurotransmitter kinetics, timings and intensity of stimulation. Inhibition delay is found to induce coherent oscillations over a broader range of experimental conditions than high-frequency entrainment. Inhibition delay boosts network capacity (ln2)-N-fold by stabilizing locally coherent oscillations. This work may inform novel therapeutic strategies for moderating pathological cortical oscillations.

Analytical particle measurements in an optical microflume.

In this work, microscopic particles in a fluid flow are manipulated using forces generated by a high power laser beam. The resulting manipulations on the particles are imaged using a microscope lens connected to a CCD camera. Differential forces on particles of varying physical and chemical composition have been measured. The goal is to measure the optical forces on a diverse range of particles and catalog the associated chemical and physical differences to understand which properties and mechanisms result in the largest force differentials. Using these measurements our aim is to better understand differences between similar microspheres in terms of size, morphology, or chemical composition. Particles of the same size, but different composition show large variations in optical pressure forces and are easily discernable in the present analytical system. In addition, we have demonstrated the ability to differentiate a 70 nm size difference between two NIST precision size standard polystyrene microspheres, corresponding to a 2.0 pN difference in optical force. Lastly, the instrument was used to measure differences between biological samples of similar size, demonstrating the ability to make precise analytical measurements on microorganism samples.

Regenerable tethered bilayer lipid membrane arrays for multiplexed label-free analysis of lipid-protein interactions on poly(dimethylsiloxane) microchips using SPR imaging.

We report a microfabrication approach to generate well-defined, addressable, and regenerable lipid membrane arrays in poly(dimethylsiloxane) (PDMS) microchips for label-free analysis of lipid-protein interactions with surface plasmon resonance imaging (SPRi). The multiplexed detection is demonstrated with a tethered bilayer membrane array built in parallel microchannels. These channels allow multiple measurements to be carried out simultaneously, showing low deviations for element-to-element variation in quantifiable signal. Lipid-conjugated receptors were utilized as model systems for protein binding analysis, and the feasibility of regenerating the tethering sublayer after binding was investigated. The results show that the lipid membrane can be removed effectively by nonionic surfactant Triton X-100. The small variance in SPR signal for the buildup process, i.e., <4% RSD for 3 cycles of detection, removal, and regeneration, indicates the sensing interface is highly reproducible. A calibration curve was obtained for cholera toxin using the monosialoganglioside (GM1) receptor, displaying a linear relationship in the 25 to 175 microg/mL range with a limit of detection of 260 nM. In addition, interaction of a phosphatidylinositol (PIP) with its binding protein and biotin/avidin interactions were employed for array measurements. To further enhance the SPR detection signal, a layer-by-layer amplification strategy was demonstrated that uses biotinylated antibody, NeutrAvidin and biotinylated anti-avidin, and the signal for protein binding on the membrane increased by 400%. The tethered membrane array technology, in combination with SPRi, offers an attractive platform for studies of membrane proteins, and can also find a range of applications for rapid screening of drug candidates interacting with proteins embedded in the near-native environment.

Cascade optical chromatography for sample fractionation.

Optical chromatography involves the elegant combination of opposing optical and fluid drag forces on colloidal samples within microfluidic environments to both measure analytical differences and fractionate injected samples. Particles that encounter the focused laser beam are trapped axially along the beam and are pushed upstream from the laser focal point to rest at a point where the optical and fluid forces on the particle balance. In our recent devices particles are pushed into a region of lower microfluidic flow, where they can be retained and fractionated. Because optical and fluid forces on a particle are sensitive to differences in the physical and chemical properties of a sample, separations are possible. An optical chromatography beam focused to completely fill a fluid channel is operated as an optically tunable filter for the separation of inorganic, polymeric, and biological particle samples. We demonstrate this technique coupled with an advanced microfluidic platform and show how it can be used as an effective method to fractionate particles from an injected multicomponent sample. Our advanced three-stage microfluidic design accommodates three lasers simultaneously to effectively create a sequential cascade optical chromatographic separation system.

Characterizing stability properties of supported bilayer membranes on nanoglassified substrates using surface plasmon resonance.

Supported bilayer membranes (SBMs) formed on solid substrates, in particular glass, provide an ideal cell mimicking model system that has been found to be highly useful for biosensing applications. Although the stability of the membrane structures is known to determine the applicability, the subject has not been extensively investigated, largely because of the lack of convenient methods to monitor changes of membrane properties on glass in real time. This work reports the evaluation of the stability properties of a series of SBMs against chemical and air damage by use of surface plasmon resonance spectroscopy and nanoglassified gold substrates. Seven SBMs composed of phosphatidylcholine and DOPC+, including single-component, mixed, protein-reinforced SBMs (rSBMs) and protein-tethered bilayer membranes (ptBLMs), are studied. The stability properties under various conditions, especially the effects of surfactants, organic solvents, and dehydration damage on the bilayers, are compared. PC membranes are found to be easily removed from the glassy surfaces using relatively low concentrations of the surfactants, while DOPC+ is markedly more stable toward nonionic surfactant. DOPC+ membranes also demonstrated remarkable air stability while PC films exhibited considerable damage from dehydration. Doping of cholesterol does not improve PC's stability against SDS and Triton but changes the lipid membrane packing enough to protect against dehydration damage. Although rSBMs and ptBLMs improve air stability to a certain degree, they are still quite susceptible to significant damage/removal from ionic and nonionic surfactants at lower concentrations. Overall, DOPC+ has noted higher stability on glass, likely due to the favorable electrostatic interaction between the silicate surface and the lipid headgroup, making it a good candidate for application. Nanoglassy SPR proves to be an attractive platform capable of rapidly screening film stability in real-time, providing critical information for future work using supported membranes for sensing applications.

Surface plasmon resonance study of protein-carbohydrate interactions using biotinylated sialosides.

Lectins are carbohydrate binding proteins found in plants, animals, and microorganisms. They serve as important models for understanding protein-carbohydrate interactions at the molecular level. We report here the fabrication of a novel sensing interface of biotinylated sialosides to probe lectin-carbohydrate interactions using surface plasmon resonance spectroscopy (SPR). The attachment of carbohydrates to the surface using biotin-NeutrAvidin interactions and the implementation of an inert hydrophilic hexaethylene glycol spacer (HEG) between the biotin and the carbohydrate result in a well-defined interface, enabling desired orientational flexibility and enhanced access of binding partners. The specificity and sensitivity of lectin binding were characterized using Sambucus nigra agglutinin (SNA) and other lectins including Maackia amurensis lectin (MAL), concanavalin A (Con A), and wheat germ agglutinin (WGA). The results indicate that alpha2,6-linked sialosides exhibit high binding affinity to SNA, while alteration in sialyl linkage and terminal sialic acid structure compromises the affinity by a varied degree. Quantitative analysis yields an equilibrium dissociation constant (KD) of 777 +/- 93 nM for SNA binding to Neu5Ac alpha2,6-LHEB. Transient SPR kinetics confirms the K D value from the equilibrium binding studies. A linear relationship was obtained in the 10-100 microg/mL range with limit of detection of approximately 50 nM. Weak interactions with MAL, Con A, and WGA were also quantified. The control experiment with bovine serum albumin indicates that nonspecific interaction on this surface is insignificant over the concentration range studied. Multiple experiments can be performed on the same substrate using a glycine stripping buffer, which selectively regenerates the surface without damaging the sialoside or the biotin-NeutrAvidin interface. This surface design retains a high degree of native affinity for the carbohydrate motifs, allowing distinction of sialyl linkages and investigation pertaining to the effect of functional group on binding efficiency. It could be easily modified to identify and quantify binding patterns of any low-affinity biologically relevant systems, opening new avenues for probing carbohydrate-protein interactions in real time.

Microfluidic fabrication of addressable tethered lipid bilayer arrays and optimization using SPR with silane-derivatized nanoglassy substrates.

We report the microfluidic fabrication of robust and fluid tethered bilayer arrays within a poly(dimethylsiloxane) (PDMS) chip, and demonstrate its addressability and biosensing by incorporating the GM1 receptor into the bilayer framework for detection of cholera toxin. Rapid optimization of the experimental conditions is achieved by using nanoglassified surfaces in combination with surface plasmon resonance. The ultrathin glassy film on gold mimics glass surfaces employed in microfluidics, allowing real-time monitoring of multiple assembly steps and therefore permitting rapid prototyping of microfluidic arrays. The tethered bilayer array utilizes a covalently immobilized biotinylated protein for generation of well-defined capture zones where a streptavidin link is employed for the immobilization of biotinylated vesicles. Fusion of captured vesicles is accomplished using a concentrated PEG solution, and the lateral diffusion of the tethered bilayer membrane is characterized by fluorescence recovery after photobleaching methods. The tethered membrane arrays demonstrate marked stability and high mobility, which provide an ideal host environment for membrane-associated proteins and open new avenues for high-throughput analysis of these proteins.