Molecular Crowding and a Minimal Footprint at a Gold Nanoparticle Support Stabilize Glucose Oxidase and Boost Its Activity.

Enzymes conjugated to nanomaterials are used in the design of various biotechnologies. In the development of biosensors, surface modifications with the enzyme glucose oxidase (GOx) serve to aid the detection of blood glucose. In order to optimize sensor effectiveness, the enzyme tertiary structure needs to be preserved upon immobilization to retain the enzyme's catalytic activity. Because of the nature of GOx, it suffers from a tendency to denature when immobilized at a solid surface; hence, methods to optimize enzyme stability are of great importance. Here, we introduce the study of the interaction of GOx to the highly curved surface of 20 nm gold nanoparticles (AuNP) with an absorbed monolayer coating of enzyme as determined by flocculation assays and quantification of immobilized GOx at the nanoparticle surface. Enzyme crowding was determined by comparing the number of enzymes that bind to how many can physically fit. These measurements show how placing a monolayer of enzyme where the enzyme spreads thin at the AuNP surface still provides stable catalytic performance of up to 14 days compared to enzymes free in solution. Moreover, by the increasing enzyme density via increasing the amount of GOx present in solution during the GOx/AuNP conjugation step creates a molecularly crowded environment at the highly curved nanoparticle surface. This limits the size of the enzyme footprint for attachment and shows that the activity per enzyme can be enhanced up to 300%. This is of great importance for implementing stable and sensitive sensor technologies that are constructed by enzyme-based nanoparticle scaffolds. Here, we show by using the conditions that maintain GOx structure and function when limiting the enzyme coating to an ultrathin layer, the design and construction of an ultrafast responding diagnostic sensor technology for glucose can be achieved, which is crucial for monitoring rapid fluctuations of, for instance, glucose in the brain.

Counting the Number of Glutamate Molecules in Single Synaptic Vesicles.

Analytical tools for quantitative measurements of glutamate, the principal excitatory neurotransmitter in the brain, are lacking. Here, we introduce a new enzyme-based amperometric sensor technique for the counting of glutamate molecules stored inside single synaptic vesicles. In this method, an ultra-fast enzyme-based glutamate sensor is placed into a solution of isolated synaptic vesicles, which stochastically rupture at the sensor surface in a potential-dependent manner at a constant negative potential. The continuous amperometric signals are sampled at high speed (10 kHz) to record sub-millisecond spikes, which represent glutamate release from single vesicles that burst open. Glutamate quantification is achieved by a calibration curve that is based on measurements of glutamate release from vesicles pre-filled with various glutamate concentrations. Our measurements show that an isolated single synaptic vesicle encapsulates about 8000 glutamate molecules and is comparable to the measured exocytotic quantal glutamate release in amperometric glutamate sensing in the nucleus accumbens of mouse brain tissue. Hence, this new methodology introduces the means to quantify ultra-small amounts of glutamate and to study synaptic vesicle physiology, pathogenesis, and drug treatments for neuronal disorders where glutamate is involved.

The evidence for open and closed exocytosis as the primary release mechanism.

Exocytosis is the fundamental process by which cells communicate with each other. The events that lead up to the fusion of a vesicle loaded with chemical messenger with the cell membrane were the subject of a Nobel Prize in 2013. However, the processes occurring after the initial formation of a fusion pore are very much still in debate. The release of chemical messenger has traditionally been thought to occur through full distention of the vesicle membrane, hence assuming exocytosis to be all or none. In contrast to the all or none hypothesis, here we discuss the evidence that during exocytosis the vesicle-membrane pore opens to release only a portion of the transmitter content during exocytosis and then close again. This open and closed exocytosis is distinct from kiss-and-run exocytosis, in that it appears to be the main content released during regular exocytosis. The evidence for this partial release via open and closed exocytosis is presented considering primarily the quantitative evidence obtained with amperometry.

Amperometry methods for monitoring vesicular quantal size and regulation of exocytosis release.

Chemical signaling strength during intercellular communication can be regulated by secretory cells through controlling the amount of signaling molecules that are released from a secretory vesicle during the exocytosis process. In addition, the chemical signal can also be influenced by the amount of neurotransmitters that is accumulated and stored inside the secretory vesicle compartment. Here, we present the development of analytical methodologies and cell model systems that have been applied in neuroscience research for gaining better insights into the biophysics and the molecular mechanisms, which are involved in the regulatory aspects of the exocytosis machinery affecting the output signal of chemical transmission at neuronal and neuroendocrine cells.

Counting the number of enzymes immobilized onto a nanoparticle-coated electrode.

To immobilize enzymes at the surface of a nanoparticle-based electrochemical sensor is a common method to construct biosensors for non-electroactive analytes. Studying the interactions between the enzymes and nanoparticle support is of great importance in optimizing the conditions for biosensor design. This can be achieved by using a combination of analytical methods to carefully characterize the enzyme nanoparticle coating at the sensor surface while studying the optimal conditions for enzyme immobilization. From this analytical approach, it was found that controlling the enzyme coverage to a monolayer was a key factor to significantly improve the temporal resolution of biosensors. However, these characterization methods involve both tedious methodologies and working with toxic cyanide solutions. Here we introduce a new analytical method that allows direct quantification of the number of immobilized enzymes (glucose oxidase) at the surface of a gold nanoparticle coated glassy carbon electrode. This was achieved by exploiting an electrochemical stripping method for the direct quantification of the density and size of gold nanoparticles coating the electrode surface and combining this information with quantification of fluorophore-labeled enzymes bound to the sensor surface after stripping off their nanoparticle support. This method is both significantly much faster compared to previously reported methods and with the advantage that this method presented is non-toxic. Graphical abstract A new analytical method for direct quantification of the number of enzymes immobilized at the surface of gold nanoparticles covering a glassy carbon electrode using anodic stripping and fluorimetry.

Lithographic Microfabrication of a 16-Electrode Array on a Probe Tip for High Spatial Resolution Electrochemical Localization of Exocytosis.

We report the lithographic microfabrication of a movable thin film microelectrode array (MEA) probe consisting of 16 platinum band electrodes placed on top of a supporting borosilicate glass substrate. These 1.2 µm wide electrodes were tightly packed and positioned parallel in two opposite rows within a 20 µm × 25 µm square area and with a distance less than 10 µm from the edge of the glass substrate. We demonstrate the ability to control and place the probe in close proximity to the surface of adherent bovine chromaffin cells and to amperometrically record single exocytosis release events with high spatiotemporal resolution. The two-dimensional position of single exocytotic events occurring in the center gap area separating the two rows of MEA band electrodes and that were codetected by electrodes in both rows was determined by analysis of the fractional detection of catecholamine released between electrodes and exploiting random walk simulations. Hence, two-dimensional electrochemical imaging recording of exocytosis release between the electrodes within this area was achieved. Similarly, by modeling the current spikes codetected by parallel adjacent band electrodes positioned in the same electrode row, a one-dimensional imaging of exocytosis with submicrometer resolution was accomplished within the area. The one- and two-dimensional electrochemical imaging using the MEA probe allowed for high spatial resolution of exocytosis activity and revealed heterogeneous release of catecholamine at the chromaffin cell surface.

Excited Fluorophores Enhance the Opening of Vesicles at Electrode Surfaces in Vesicle Electrochemical Cytometry.

Electrochemical cytometry is a method developed recently to determine the content of an individual cell vesicle. The mechanism of vesicle rupture at the electrode surface involves the formation of a pore at the interface between a vesicle and the electrode through electroporation, which leads to the release and oxidation of the vesicle's chemical cargo. We have manipulated the membrane properties using excited fluorophores conjugated to lipids, which appears to make the membrane more susceptible to electroporation. We propose that by having excited fluorophores in close contact with the membrane, membrane lipids (and perhaps proteins) are oxidized upon production of reactive oxygen species, which then leads to changes in membrane properties and the formation of water defects. This is supported by experiments in which the fluorophores were placed on the lipid tail instead of the headgroup, which leads to a more rapid onset of vesicle opening. Additionally, application of DMSO to the vesicles, which increases the membrane area per lipid, and decreasing the membrane thickness result in the same enhancement in vesicle opening, which confirms the mechanism of vesicle opening with excited fluorophores in the membrane. Light-induced manipulation of membrane vesicle pore opening might be an attractive means of controlling cell activity and exocytosis. Additionally, our data confirm that in experiments in which cells or vesicle membranes are labeled for fluorescence monitoring, the properties of the excited membrane change substantially.

Characterizing the catecholamine content of single mammalian vesicles by collision-adsorption events at an electrode.

We present the electrochemical response to single adrenal chromaffin vesicles filled with catecholamine hormones as they are adsorbed and rupture on a 33 µm diameter disk-shaped carbon electrode. The vesicles adsorb onto the electrode surface and sequentially spread out over the electrode surface, trapping their contents against the electrode. These contents are then oxidized, and a current (or amperometric) peak results from each vesicle that bursts. A large number of current transients associated with rupture of single vesicles (86%) are observed under the experimental conditions used, allowing us to quantify the vesicular catecholamine content.

Coimmobilization of acetylcholinesterase and choline oxidase on gold nanoparticles: stoichiometry, activity, and reaction efficiency.

Hybrid structures constructed from biomolecules and nanomaterials have been used in catalysis and bioanalytical applications. In the design of many chemically selective biosensors, enzymes conjugated to nanoparticles or carbon nanotubes have been used in functionalization of the sensor surface for enhancement of the biosensor functionality and sensitivity. The conditions for the enzyme:nanomaterial conjugation should be optimized to retain maximal enzyme activity, and biosensor effectiveness. This is important as the tertiary structure of the enzyme is often altered when immobilized and can significantly alter the enzyme catalytic activity. Here we show that characterization of a two-enzyme:gold nanoparticle (AuNP) conjugate stoichiometry and activity can be used to gauge the effectiveness of acetylcholine detection by acetylcholine esterase (AChE) and choline oxidase (ChO). This was done by using an analytical approach to quantify the number of enzymes bound per AuNP and monitor the retained enzyme activity after the enzyme:AuNP synthesis. We found that the amount of immobilized enzymes differs from what would be expected from bulk solution chemistry. This analysis was further used to determine the optimal ratio of AChE:ChO added at synthesis to achieve optimum sequential enzyme activity for the enzyme:AuNP conjugates, and reaction efficiencies of greater than 70%. We here show that the knowledge of the conjugate stoichiometry and retained enzyme activity can lead to more efficient detection of acetylcholine by controlling the AChE:ChO ratio bound to the gold nanoparticle material. This approach of optimizing enzyme gold nanoparticle conjugates should be of great importance in the architecture of enzyme nanoparticle based biosensors to retain optimal sensor sensitivity.

Artificial Cells for Dissecting Exocytosis.

The fusion of vesicles and exocytosis release of neurotransmitters into the extracellular space for detection and chemical signal decoding by neighboring cells is the key process in neuronal communication. It is important to understand what regulates exocytosis because the amount of neurotransmitters released into the synaptic cleft has a direct impact on brain function such as cognition learning and memory as well as on brain malfunctions. Much success in molecular biology can be credited for the existence of simplified model systems. Therefore, for gaining deeper insights into the details of exocytosis and what controls vesicle-mediated neurotransmission, functional artificial cells for exocytosis have been developed that can be used for studying various biophysical aspects and roles of molecules affecting exocytosis, which is difficult to study in living cells. Here, we describe the design and fabrication of specific artificial cell models and how chemical measurements at these cells can be implemented for probing dynamics of the exocytosis fusion pore and its effect on the regulation of neurochemical release. We introduce bottom-up synthetic methods for constructing model cells using protein-free giant unilamellar vesicles (GUV) as starting material, which allows further tuning of molecular complexity in a manner that is not possible in living cells and therefore can be used for dissecting the role of essential molecular components affecting the exocytosis process. The experimental setup uses microscopy video recording, micromanipulation and microelectroinjection techniques, and amperometry detection to study neurotransmitter release from these cells mimicking exocytosis.

Carbon-ring microelectrode arrays for electrochemical imaging of single cell exocytosis: fabrication and characterization.

Fabrication of carbon microelectrode arrays, with up to 15 electrodes in total tips as small as 10-50 µm, is presented. The support structures of microelectrodes were obtained by pulling multiple quartz capillaries together to form hollow capillary arrays before carbon deposition. Carbon ring microelectrodes were deposited by pyrolysis of acetylene in the lumen of these quartz capillary arrays. Each carbon deposited array tip was filled with epoxy, followed by beveling of the tip of the array to form a deposited carbon-ring microelectrode array (CRMA). Both the number of the microelectrodes in the array and the tip size are independently tunable. These CRMAs have been characterized using scanning electron microscopy, energy dispersive X-ray spectroscopy, and electrogenerated chemiluminescence. Additionally, the electrochemical properties were investigated with steady-state voltammetry. In order to demonstrate the utility of these fabricated microelectrodes in neurochemistry, CRMAs containing eight microring electrodes were used for electrochemical monitoring of exocytotic events from single PC12 cells. Subcellular temporal heterogeneities in exocytosis (i.e. cold spots vs hot spots) were successfully detected with the CRMAs.

Analytical tools to monitor exocytosis: a focus on new fluorescent probes and methods.

A great deal of research has been focused on unraveling the processes governing the exocytotic pathway and the extent of release during the process. Arguments abound for and against both the occurrence and significance of full release during exocytosis and partial release including kiss-and-run events. Several optical methods to directly observe the exocytosis process have been developed and here we focus on fluorescence methods and probes for this work. Although fluorescence imaging has been used for cell experiments for decades, in the last two decades a plethora of new approaches have arrived on the scene. These include application of new microscopy techniques, like total internal reflectance and stimulated emission depletion that are offering new ways to circumvent the limits of far field microscopy with a diffraction limit of 200 nm, and allow tracking of single synaptic vesicles. For selective imaging of synaptic vesicles the introduction of methods to stain the vesicular compartment has involved developing probes of the vesicular membrane and intravesicular solution, nanoparticle quantum dots that can be observed during exocytosis but not via the fusion pore, and fluorescent false neurotransmitters.

Steady-state electrochemical determination of lipidic nanotube diameter utilizing an artificial cell model.

By exploiting the capabilities of steady-state electrochemical measurements, we have measured the inner diameter of a lipid nanotube using Fick's first law of diffusion in conjunction with an imposed linear concentration gradient of electroactive molecules over the length of the nanotube. Fick's law has been used in this way to provide a direct relationship between the nanotube diameter and the measurable experimental parameters Deltai (change in current) and nanotube length. Catechol was used to determine the Deltai attributed to its flux out of the nanotube. Comparing the nanotube diameter as a function of nanotube length revealed that membrane elastic energy was playing an important role in determining the size of the nanotube and was different when the tube was connected to either end of two vesicles or to a vesicle on one end and a pipet tip on the other. We assume that repulsive interaction between neck regions can be used to explain the trends observed. This theoretical approach based on elastic energy considerations provides a qualitative description consistent with experimental data.

Electrochemical probes for detection and analysis of exocytosis and vesicles.

Unraveling the mechanistic details of neurotransmitter exocytosis is arguably among the most important molecular problems in neuroscience today. Investigations at single cells, particularly with electrochemical methods, have given unique chemical and biological insight into this process at the fundamental level. The rapid response time (submillisecond) of microelectrodes makes them well suited for monitoring the dynamic process of exocytosis. We review here recent developments in electrochemical techniques to spatially and simultaneously detect exocytosis across a single cell and to measure the transmitter content of single vesicles removed from cells. The former method is used to demonstrate dynamic heterogeneity in release across a cell, and in the latter work comparison is made between vesicle content and release to conclude that only a fraction of the transmitter is released during full exocytosis.

Analytical approaches to investigate transmitter content and release from single secretory vesicles.

The vesicle serves as the primary intracellular unit for the highly efficient storage and release of chemical messengers triggered during signaling processes in the nervous system. This review highlights conventional and emerging analytical methods that have used microscopy, electrochemistry, and spectroscopy to resolve the location, time course, and quantal content characteristics of neurotransmitter release. Particular focus is on the investigation of the synaptic vesicle and its involvement in the fundamental molecular mechanisms of cell communication.

Electrochemistry of Single-Vesicle Events.

Neuronal transmission relies on electrical signals and the transfer of chemical signals from one neuron to another. Chemical messages are transmitted from presynaptic neurons to neighboring neurons through the triggered fusion of neurotransmitter-filled vesicles with the cell plasma membrane. This process, known as exocytosis, involves the rapid release of neurotransmitter solutions that are detected with high affinity by the postsynaptic neuron. The type and number of neurotransmitters released and the frequency of vesicular events govern brain functions such as cognition, decision making, learning, and memory. Therefore, to understand neurotransmitters and neuronal function, analytical tools capable of quantitative and chemically selective detection of neurotransmitters with high spatiotemporal resolution are needed. Electrochemistry offers powerful techniques that are sufficiently rapid to allow for the detection of exocytosis activity and provides quantitative measurements of vesicle neurotransmitter content and neurotransmitter release from individual vesicle events. In this review, we provide an overview of the most commonly used electrochemical methods for monitoring single-vesicle events, including recent developments and what is needed for future research.

Generation of interconnected vesicles in a liposomal cell model.

We introduce an experimental method based upon a glass micropipette microinjection technique for generating a multitude of interconnected vesicles (IVs) in the interior of a single giant unilamellar phospholipid vesicle (GUV) serving as a cell model system. The GUV membrane, consisting of a mixture of soybean polar lipid extract and anionic phosphatidylserine, is adhered to a multilamellar lipid vesicle that functions as a lipid reservoir. Continuous IV formation was achieved by bringing a micropipette in direct contact with the outer GUV surface and subjecting it to a localized stream of a Ca2+ solution from the micropipette tip. IVs are rapidly and sequentially generated and inserted into the GUV interior and encapsulate portions of the micropipette fluid content. The IVs remain connected to the GUV membrane and are interlinked by short lipid nanotubes and resemble beads on a string. The vesicle chain-growth from the GUV membrane is maintained for as long as there is the supply of membrane material and Ca2+ solution, and the size of the individual IVs is controlled by the diameter of the micropipette tip. We also demonstrate that the IVs can be co-loaded with high concentrations of neurotransmitter and protein molecules and displaying a steep calcium ion concentration gradient across the membrane. These characteristics are analogous to native secretory vesicles and could, therefore, serve as a model system for studying secretory mechanisms in biological systems.

Ultrafast Glutamate Biosensor Recordings in Brain Slices Reveal Complex Single Exocytosis Transients.

Neuronal communication relies on vesicular neurotransmitter release from signaling neurons and detection of these molecules by neighboring neurons. Glutamate, the main excitatory neurotransmitter in the mammalian brain, is involved in nearly all brain functions. However, glutamate has suffered from detection schemes that lack temporal and spatial resolution allowed by electrochemistry. Here we show an amperometric, novel, ultrafast enzyme-based nanoparticle modified sensor, measuring random bursts of hundreds to thousands of rapid spontaneous glutamate exocytotic release events at approximately 30 Hz frequency in the nucleus accumbens of rodent brain slices. Characterizing these single submillisecond exocytosis events revealed a great diversity in spike shape characteristics and size of quantal release, suggesting variability in fusion pore dynamics controlling the glutamate release by cells in this brain region. Hence, this novel biosensor allows recording of rapid single glutamate exocytosis events in the brain tissue and offers insight on regulatory aspects of exocytotic glutamate release, which is critical to understanding of brain glutamate function and dysfunction.

Positioning lipid membrane domains in giant vesicles by micro-organization of aqueous cytoplasm mimic.

We report localization of lipid membrane microdomains to specific "poles" of asymmetric giant vesicles (GVs) in response to local internal composition. Interior aqueous microdomains were generated in a simple model cytoplasm composed of a poly(ethyleneglycol) (PEG)/dextran aqueous two-phase system (ATPS) encapsulated in the vesicles. The GV membrane composition used here was a modification of a DOPC/DPPC/cholesterol mixture known to form micrometer-scale liquid ordered and liquid disordered domains; we added lipids with PEG 2000 Da-modified headgroups. Osmotically induced budding of the ATPS-containing GVs led to structures where the PEG-rich and dextran-rich interior aqueous phases were in contact with different regions of the vesicle membrane. Liquid ordered (L o) membrane domains rich in PEG-terminated lipids preferentially coated the PEG-rich aqueous phase vesicle "body", while coexisting liquid disordered (L d) membrane domains coated the dextran-rich aqueous phase "bud". Membrane domain positioning resulted from interactions between lipid headgroups and the interior aqueous polymer solutions, e.g., PEGylated headgroups with PEG and dextran polymers. Heating resulted first in patchy membranes where L o and L d domains no longer showed any preference for coating the PEG-rich vs dextran-rich interior aqueous volumes, and eventually complete lipid mixing. Upon cooling lipid domains again coated their preferred interior aqueous microvolume. This work shows that nonspecific interactions between interior aqueous contents and the membrane that encapsulates them can drive local chemical heterogeneity, and offers a primitive experimental model for membrane and cytoplasmic polarity in biological cells.

Budding and asymmetric protein microcompartmentation in giant vesicles containing two aqueous phases.

We report the effect of external osmolarity on giant lipid vesicles containing an aqueous two-phase system (ATPS GVs). The ATPS, which is comprised of poly(ethyleneglycol) [PEG], dextran, and water, serves as a primitive model of the macromolecularly crowded environment of the cytoplasm. Coexisting PEG-rich and dextran-rich aqueous phases provide chemically dissimilar microenvironments, enabling local differences in protein concentration to be maintained within single ATPS GVs. The degree of biomolecule microcompartmentation can be increased by exposing the ATPS GVs to a hypertonic external solution, which draws water out of the vesicles, concentrating the polymers. Enrichment of a protein, soybean agglutinin, in the dextran-rich phase improves from 2.3-fold to 10-fold with an increase in external osmolarity from 100 to 200 mmol/kg. In some cases, budding occurs, with the bud(s) formed by partial expulsion of one of the two polymer-rich aqueous phases. Budding results in asymmetry in the internal polymer and biomolecule composition, giving rise to polarity in these primitive model cells. Budding is observed with increasing frequency as external ionic strength increases, when membrane elasticity permits, and can be reversed by decreasing external osmolarity. We note that the random symmetry-breaking induced by simple osmotic shrinkage resulted in polarity in both the structure and internal protein distribution in these primitive model cells. Budding in ATPS-containing GVs thus offers an experimental model system for investigating the effects of biochemical asymmetry on the length scale of single cells.