formation-biomembrane-microarrays-with-squeegee-based-assembly

Formation of Biomembrane Microarrays with a Squeegee-based Assembly Method

Lipid bilayer membranes form the plasma membranes of cells and define the boundaries of subcellular organelles. In nature, these membranes are ...

characterizing-single-molecule-conformational-changes-under-shear

Characterizing Single-Molecule Conformational Changes Under Shear Flow with Fluorescence Microscopy

Single-molecule behavior under mechanical perturbation has been characterized widely to understand many biological processes. However, methods such as ...

University of Minnesota

Lehigh University

We have amperometrically measured dopamine release from rat pheochromocytoma cells (PC12 cells) in high osmolarity conditions with and without L-3,4-dihydroxyphenylalanine (L-DOPA) treatment. We observe an increase in the number of release events displaying a prespike feature or "foot" when the cells are stimulated in high osmolarity saline. We also see an increase in foot area and duration when cells are stimulated in high osmolarity saline, or high osmolarity saline subsequent to incubation with the dopamine precursor L-DOPA in isotonic saline, which serves to increase the vesicle size. The data suggest that membrane biophysics are an important component in defining the rate, duration and amount of neurotransmitter release via the fusion pore.

High osmolarity and L-DOPA augment release via the fusion pore in PC12 cells.

We have used amperometric measurements in a model system consisting of two liposomes connected with a membrane nanotube to monitor catechol release during artificial exocytosis and thereby to elucidate the effect of small-chain alcohols on this dynamic membrane process. To determine the rate of membrane shape change, catechol release during membrane distention was monitored amperometrically, and the presence of alcohols in the buffer was shown to accelerate the membrane distention process in a concentration-dependent manner. Compression isotherms for the same lipid composition in the absence and presence of ethanol and 1-propanol were measured to determine how these short-chain alcohols affect the lipid packing in monolayers. The isotherms show a marked decrease in lipid packing density that is dependent on the particular alcohol and its concentration. Comparison of the electrochemical and isotherm results suggests a correlation between decreasing lipid packing density and increasing rates of membrane shape change.

Short-chain alcohols promote accelerated membrane distention in a dynamic liposome model of exocytosis.

Carbon-fiber microelectrochemical methods were utilized in this study to measure individual exocytotic events of secretion of serotonin and histamine from washed rabbit platelets. The quantal release of serotonin was quantitatively characterized with a delta-granule serotonin concentration of 0.6 M and secretion time course of 7 ms. Additionally, extracellular osmolarity influences quantal size, causing quantal size increases under hypotonic conditions, presumably due to the influx of cytosolic serotonin into the halo region of the delta-granules.

Quantitative and real-time detection of secretion of chemical messengers from individual platelets.

The size-dependent chemical and physical properties of nanoparticles inspire the design of unique assays and the use of new detection schemes while also offering the opportunity to vastly improve the results achieved when using traditional signal transduction methods. Herein, the most commonly exploited nanoparticle amplification schemes are organized and reviewed on the basis of the detection methods used to monitor the nanoparticle property of interest. The topics covered include the improved signal photostability and brightness of semiconductor quantum dots, the increased extinction coefficient of noble metal nanoparticles, the advantages of having a magnetic label on individual target molecules to facilitate separation, the multiplexing that is enabled with 'barcoded' nanoparticles, and the greatly amplified signals that can be achieved on the basis of conductivity changes, generated current, or simply by adding a 'massive' nanoparticle onto a small molecule target. Common approaches emerge among different nanoparticle materials and detection schemes, and it is also clear that there is still significant opportunity to use nanoparticles in as-yet-unimagined ways to further improve assay and sensor limits of detection.

Using nanoparticles to push the limits of detection.

Integration of solid-state biosensors and lipid bilayer membranes is important for membrane protein research and drug discovery. In these sensors, it is critical that the solid-state sensing material does not have adverse effects on the conformation or functionality of membrane-bound molecules. In this work, pore-spanning lipid membranes are formed over an array of periodic nanopores in free-standing gold films for surface plasmon resonance (SPR) kinetic binding assays. The ability to perform kinetic assays with a transmembrane protein is demonstrated with α-hemolysin (α-HL). The incorporation of α-HL into the membrane followed by specific antibody binding (anti-α-HL) red-shifts the plasmon resonance of the gold nanopore array, which is optically monitored in real time. Subsequent fluorescence imaging reveals that the antibodies primarily bind in nanopore regions, indicating that α-HL incorporation preferentially occurs into areas of pore-spanning lipid membranes.

Membrane protein biosensing with plasmonic nanopore arrays and pore-spanning lipid membranes.

Questa prospettiva fornisce una panoramica dei recenti sviluppi in superficie-enhanced Raman scattering (SERS) per biosensori. Attenzione questa recensione su SERS articoli pubblicati negli ultimi 10 anni e per specifiche applicazioni di rilevamento biologico analiti. Schemi di biosensori SERS sia intrinseci che estrinseci sono stati impiegati per rilevare e identificare piccole molecole, gli acidi nucleici, lipidi, peptidi e proteine, come pure per la rilevazione in vivo e cellulare. Attuali tecnologie di substrato SERS insieme a una serie di avanzamenti nella chimica delle superfici, preparazione del campione, schemi di trasduzione del segnale intrinseca/estrinseca e spettroscopia Raman punta avanzata sono discussi. Il progresso contemplato nel presente documento mostra la promessa grande per adozione diffusa di biosensori SERS.

Recenti progressi in biosensori SERS.

Cette perspective donne un aper&ccedil;u des r&eacute;cents d&eacute;veloppements en Raman exalt&eacute;e de surface (SERS) de diffusion de biod&eacute;tection. Nous visons cette revue SERS documents publi&eacute;s au cours des 10 derni&egrave;res ann&eacute;es et &agrave; des applications sp&eacute;cifiques de d&eacute;tection des analytes biologiques. R&eacute;gimes de biod&eacute;tection SERS tant intrins&egrave;ques qu&#39;extrins&egrave;ques ont &eacute;t&eacute; employ&eacute;es pour d&eacute;tecter et identifier des petites mol&eacute;cules d&#39;acides nucl&eacute;iques, lipides, peptides et prot&eacute;ines, comme ainsi que pour detection in vivo et cellulaire. Les auteurs discutent les technologies actuelles de substrat SERS ainsi qu&#39;une s&eacute;rie de progr&egrave;s en chimie de surface, pr&eacute;paration des &eacute;chantillons, syst&egrave;mes de transduction de signal intrins&egrave;ques et extrins&egrave;ques et spectroscopie de Raman pointe renforc&eacute;e. Les progr&egrave;s vis&eacute;s dans les pr&eacute;sentes sont tr&egrave;s prometteur pour l&#39;adoption g&eacute;n&eacute;ralis&eacute;e de biod&eacute;tection SERS.

Progr&egrave;s r&eacute;cents en biod&eacute;tection SERS.

This perspective gives an overview of recent developments in surface-enhanced Raman scattering (SERS) for biosensing. We focus this review on SERS papers published in the last 10 years and to specific applications of detecting biological analytes. Both intrinsic and extrinsic SERS biosensing schemes have been employed to detect and identify small molecules, nucleic acids, lipids, peptides, and proteins, as well as for in vivo and cellular sensing. Current SERS substrate technologies along with a series of advancements in surface chemistry, sample preparation, intrinsic/extrinsic signal transduction schemes, and tip-enhanced Raman spectroscopy are discussed. The progress covered herein shows great promise for widespread adoption of SERS biosensing.

Recent progress in SERS biosensing.

Inexpensive, reproducible, and high-throughput fabrication of nanometric apertures in metallic films can benefit many applications in plasmonics, sensing, spectroscopy, lithography, and imaging. Here we use template-stripping to pattern periodic nanohole arrays in optically thick, smooth Ag films with a silicon template made via nanoimprint lithography. Ag is a low-cost material with good optical properties, but it suffers from poor chemical stability and biocompatibility. However, a thin silica shell encapsulating our template-stripped Ag nanoholes facilitates biosensing applications by protecting the Ag from oxidation as well as providing a robust surface that can be readily modified with a variety of biomolecules using well-established silane chemistry. The thickness of the conformal silica shell can be precisely tuned by atomic layer deposition, and a 15 nm thick silica shell can effectively prevent fluorophore quenching. The Ag nanohole arrays with silica shells can also be bonded to polydimethylsiloxane (PDMS) microfluidic channels for fluorescence imaging, formation of supported lipid bilayers, and real-time, label-free SPR sensing. Additionally, the smooth surfaces of the template-stripped Ag films enhance refractive index sensitivity compared with as-deposited, rough Ag films. Because nearly centimeter-sized nanohole arrays can be produced inexpensively without using any additional lithography, etching, or lift-off, this method can facilitate widespread applications of metallic nanohole arrays for plasmonics and biosensing.

Template-stripped smooth Ag nanohole arrays with silica shells for surface plasmon resonance biosensing.

Microarray technology has facilitated many powerful high-throughput studies in the fields of genetics and proteomics, among others. However, preparation of microarrays composed of cell-derived membranes with embedded receptors has proven difficult. Here we describe a new method for forming microarrays composed of synthetic lipid vesicles and natural cell membranes. The method is based upon assembly of vesicles and natural membranes into recessed micro- and nanowells and using a polydimethylsiloxane (PDMS) block as a "squeegee." This method is used to assemble phospholipid vesicles into arrays with micrometer and nanoscale dimensions. Native myelin and neuronal lipid raft arrays are also formed in 30 min or less. We show the natural membrane arrays can be used for sensing lipid-protein interactions by detecting cholera toxin binding to ganglioside GM1 in neuronal lipid rafts. In multicomponent arrays myelin can be distinguished from neuronal rafts by antibody binding to cell-specific surface antigens. Finally, myelin arrays formed in gold nanowells are used for surface plasmon resonance sensing. This assembly approach is simple, broadly applicable, and opens up new avenues of research not easily accomplished with standard microarray technology.

Facile assembly of micro- and nanoarrays for sensing with natural cell membranes.

Multiple sclerosis is a progressive neurological disorder that results in the degradation of myelin sheaths that insulate axons in the central nervous system. Therefore promotion of myelin repair is a major thrust of multiple sclerosis treatment research. Two mouse monoclonal natural autoantibodies, O1 and O4, promote myelin repair in several mouse models of multiple sclerosis. Natural autoantibodies are generally polyreactive and predominantly of the IgM isotype. The prevailing paradigm is that because they are polyreactive, these antibodies bind antigens with low affinities. Despite their wide use in neuroscience and glial cell research, however, the affinities and kinetic constants of O1 and O4 antibodies have not been measured to date. In this work, we developed a membrane biosensing platform based on surface plasmon resonance in gold nanohole arrays with a series of surface modification techniques to form myelin-mimicking lipid bilayer membranes to measure both the association and dissociation rate constants for O1 and O4 antibodies binding to their myelin lipid antigens. The ratio of rate constants shows that O1 and O4 bind to galactocerebroside and sulfated galactocerebroside, respectively, with unusually small apparent dissociation constants (K(D) ≈ 0.9 nM) for natural autoantibodies. This is approximately one to 2 orders of magnitude lower than typically observed for the highest affinity natural autoantibodies. We propose that the unusually high affinity of O1 and O4 to their targets in myelin contributes to the mechanism by which they signal oligodendrocytes and induce central nervous system repair.

High-affinity binding of remyelinating natural autoantibodies to myelin-mimicking lipid bilayers revealed by nanohole surface plasmon resonance.

While atomic layer deposition (ALD) has been used for many years as an industrial manufacturing method for microprocessors and displays, this versatile technique is finding increased use in the emerging fields of plasmonics and nanobiotechnology. In particular, ALD coatings can modify metallic surfaces to tune their optical and plasmonic properties, to protect them against unwanted oxidation and contamination, or to create biocompatible surfaces. Furthermore, ALD is unique among thin-film deposition techniques in its ability to meet the processing demands for engineering nanoplasmonic devices, offering conformal deposition of dense and ultra-thin films on high-aspect-ratio nanostructures at temperatures below 100 °C. In this review, we present key features of ALD and describe how it could benefit future applications in plasmonics, nanosciences, and biotechnology.

Atomic layer deposition (ALD): A versatile technique for plasmonics and nanobiotechnology.

With recent advances in high-throughput proteomics and systems biology, there is a growing demand for new instruments that can precisely quantify a wide range of receptor-ligand binding kinetics in a high-throughput fashion. Here we demonstrate a surface plasmon resonance (SPR) imaging spectroscopy instrument capable of simultaneously extracting binding kinetics and affinities from 50 parallel microfluidic channels. The instrument utilizes large-area (~ cm(2)) metallic nanohole arrays as SPR sensing substrates and combines a broadband light source, a high-resolution imaging spectrometer and a low-noise CCD camera to extract spectral information from every channel in real time with a refractive index resolution of 7.7 × 10(-6) refractive index units. To demonstrate the utility of our instrument for quantifying a wide range of biomolecular interactions, each parallel microfluidic channel is coated with a biomimetic supported lipid membrane containing ganglioside (GM1) receptors. The binding kinetics of cholera toxin b (CTX-b) to GM1 are then measured in a single experiment from 50 channels. By combining the highly parallel microfluidic device with large-area periodic nanohole array chips, our SPR imaging spectrometer system enables high-throughput, label-free, real-time SPR biosensing, and its full-spectral imaging capability combined with nanohole arrays could enable integration of SPR imaging with concurrent surface-enhanced Raman spectroscopy.

Real-time full-spectral imaging and affinity measurements from 50 microfluidic channels using nanohole surface plasmon resonance.

Lipid bilayer membranes found in nature are heterogeneous mixtures of lipids and proteins. Model systems, such as supported lipid bilayers (SLBs), are often employed to simplify experimental systems while mimicking the properties of natural lipid bilayers. Here, we demonstrate a new method to form SLB arrays by first forming spherical supported lipid bilayers (SSLBs) on submicrometer-diameter SiO(2) beads. The SSLBs are then arrayed into microwells using a simple physical assembly method that requires no chemical modification of the substrate nor modification of the lipid membrane with recognition moieties. The resulting arrays have submicrometer SSLBs with 3 μm periodicity where >75% of the microwells are occupied by an individual SSLB. Because the arrays have high density, fluorescence from >1000 discrete SSLBs can be acquired with a single image capture. We show that 2-component random arrays can be formed, and we also use the arrays to determine the equilibrium dissociation constant for cholera toxin binding to ganglioside GM1. SSLB arrays are robust and are stable for at least one week in buffer.

High-density arrays of submicron spherical supported lipid bilayers.

Metallic nanopore arrays have emerged as optofluidic platforms with multifarious sensing and analytical capabilities such as label-free surface plasmon resonance (SPR) sensing of molecular binding interactions and surface-enhanced Raman spectroscopy (SERS). However, directed delivery of analytes through open nanopores using traditional methods such as external electric fields or pressure gradients still remains difficult. We demonstrate that nanopore arrays have an intrinsic ability to promote flow through them via capillary flow and evaporation. This passive "nano-drain" mechanism is utilized to concentrate biomolecules on the surface of nanopores for improved detection sensitivity or create ordered nanoscale arrays of beads and liposomes. Without using any external pump or fluidic interconnects, we can concentrate and detect the presence of less than a femtomole of streptavidin in 10 μL of sample using fluorescence imaging. Liposome nanoarrays are also prepared in less than 5 min and used to detect lipid-protein interactions. We also demonstrate label-free SPR detection of analytes using metallic nanopore arrays. This method provides a fast, simple, transportable, and small-volume platform for labeled as well as label-free plasmonic analysis while improving the detection time and sensitivity.

Nanopore-induced spontaneous concentration for optofluidic sensing and particle assembly.

Matrix molecules convey biochemical and physical guiding signals to neurons in the central nervous system (CNS) and shape the trajectory of neuronal fibers that constitute neural networks. We have developed recombinant human IgMs that bind to epitopes on neural cells, with the aim of treating neurological diseases. Here we test the hypothesis that recombinant human IgMs (rHIgM) can guide neurite outgrowth of CNS neurons. Microcontact printing was employed to pattern rHIgM12 and rHIgM22, antibodies that were bioengineered to have variable regions capable of binding to neurons or oligodendrocytes, respectively. rHIgM12 promoted neuronal attachment and guided outgrowth of neurites from hippocampal neurons. Processes from spinal neurons followed grid patterns of rHIgM12 and formed a physical network. Comparison between rHIgM12 and rHIgM22 suggested the biochemistry that facilitates anchoring the neuronal surfaces is a prerequisite for the function of IgM, and spatial properties cooperate in guiding the assembly of neuronal networks.

A patterned recombinant human IgM guides neurite outgrowth of CNS neurons.

Optical biosensors based on surface plasmon resonance (SPR) in metallic thin films are currently standard tools for measuring molecular binding kinetics and affinities - an important task for biophysical studies and pharmaceutical development. Motivated by recent progress in the design and fabrication of metallic nanostructures, such as nanoparticles or nanoholes of various shapes, researchers have been pursuing a new generation of biosensors harnessing tailored plasmonic effects in these engineered nanostructures. Nanoplasmonic devices, while demanding nanofabrication, offer tunability with respect to sensor dimension and physical properties, thereby enabling novel biological interfacing opportunities and extreme miniaturization. Here we provide an integrated overview of refractometric biosensing with nanoplasmonic devices and highlight some recent examples of nanoplasmonic sensors capable of unique functions that are difficult to accomplish with conventional SPR. For example, since the local field strength and spatial distribution can be readily tuned by varying the shape and arrangement of nanostructures, biomolecular interactions can be controlled to occur in regions of high field strength. This may improve signal-to-noise and also enable sensing a small number of molecules. Furthermore, the nanoscale plasmonic sensor elements may, in combination with nanofabrication and materials-selective surface-modifications, make it possible to merge affinity biosensing with nanofluidic liquid handling.

Promises and Challenges of Nanoplasmonic Devices for Refractometric Biosensing.

Characterization of binding kinetics and affinity between a potential drug and its receptor are key steps in the development of new drugs. Among the techniques available to determine binding affinities, surface plasmon resonance has emerged as the gold standard because it can measure binding and dissociation rates in real-time in a label-free fashion. Surface plasmon resonance is now finding applications in the characterization of molecules for treatment of neurodegenerative diseases, characterization of molecules associated with pathogenesis of neurodegenerative diseases and detection of neurodegenerative disease biomarkers. In addition it has been used in the characterization of a new class of natural autoantibodies that have therapeutic potential in a number of neurologic diseases. In this review we will introduce surface plasmon resonance and describe some applications of the technique that pertain to neurodegenerative disorders and their treatment.

ArticleTotal : 2	Year
Publication title Cited by 1	2014
Publication title Cited by 1	2020

Article	Year
High osmolarity and L-DOPA augment release via the fusion pore in PC12 cells. Chemphyschem : a European journal of chemical physics and physical chemistry\| PubMed ID: 17966970	2007
Short-chain alcohols promote accelerated membrane distention in a dynamic liposome model of exocytosis. Langmuir : the ACS journal of surfaces and colloids\| PubMed ID: 18278956	2008
Quantitative and real-time detection of secretion of chemical messengers from individual platelets. Biochemistry\| PubMed ID: 18557631	2008
Using nanoparticles to push the limits of detection. Wiley interdisciplinary reviews. Nanomedicine and nanobiotechnology\| PubMed ID: 20049794	2009
Membrane protein biosensing with plasmonic nanopore arrays and pore-spanning lipid membranes. Chemical science (Royal Society of Chemistry : 2010)\| PubMed ID: 21218136	2010
Recent progress in SERS biosensing. Physical chemistry chemical physics : PCCP\| PubMed ID: 21509385	2011
Template-stripped smooth Ag nanohole arrays with silica shells for surface plasmon resonance biosensing. ACS nano\| PubMed ID: 21770414	2011
Facile assembly of micro- and nanoarrays for sensing with natural cell membranes. ACS nano\| PubMed ID: 21842844	2011
High-affinity binding of remyelinating natural autoantibodies to myelin-mimicking lipid bilayers revealed by nanohole surface plasmon resonance. Analytical chemistry\| PubMed ID: 22762372	2012
Atomic layer deposition (ALD): A versatile technique for plasmonics and nanobiotechnology. Journal of materials research\| PubMed ID: 22865951	2012
Real-time full-spectral imaging and affinity measurements from 50 microfluidic channels using nanohole surface plasmon resonance. Lab on a chip\| PubMed ID: 22895607	2012
High-density arrays of submicron spherical supported lipid bilayers. Analytical chemistry\| PubMed ID: 22967217	2012
Nanopore-induced spontaneous concentration for optofluidic sensing and particle assembly. Analytical chemistry\| PubMed ID: 23214989	2013
A patterned recombinant human IgM guides neurite outgrowth of CNS neurons. Scientific reports\| PubMed ID: 23881231	2013
Promises and Challenges of Nanoplasmonic Devices for Refractometric Biosensing. Nanophotonics\| PubMed ID: 24159429	2013
Applications of SPR for the characterization of molecules important in the pathogenesis and treatment of neurodegenerative diseases. Expert review of neurotherapeutics\| PubMed ID: 24625008	2014

Nathan J. Wittenberg

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