Ion Channel Biosensor Research Articles

Hydrophobic Gating and 1/f Noise of the Anthrax Toxin Channel.

"Pink" or 1/f noise is a natural phenomenon omnipresent in physics, economics, astrophysics, biology, and even music and languages. In electrophysiology, the stochastic activity of a number of biological ion channels and artificial nanopores could be characterized by current noise with a 1/f power spectral density. In the anthrax toxin channel (PA63), it appears as fast voltage-independent current interruptions between conducting and nonconducting states. This behavior hampers potential development of PA63 as an ion-channel biosensor. On the bright side, the PA63 flickering represents a mesmerizing phenomenon to investigate. Notably, similar 1/f fluctuations are observed in the channel-forming components of clostridial binary C2 and iota toxins, which share functional and structural similarities with the anthrax toxin channel. Similar to PA63, they are evolved to translocate the enzymatic components of the toxins into the cytosol. Here, using high-resolution single-channel lipid bilayer experiments and all-atom molecular dynamic simulations, we suggest that the 1/f noise in PA63 occurs as a result of "hydrophobic gating" at the ϕ-clamp region, the phenomenon earlier observed in several water-filled channels "fastened" inside by the hydrophobic belts. The ϕ-clamp is a narrow "hydrophobic ring" in the PA63 lumen formed by seven or eight phenylalanine residues at position 427, conserved in the C2 and iota toxin channels, which catalyzes protein translocation. Notably, the 1/f noise remains undetected in the F427A PA63 mutant. This finding can elucidate the functional purpose of 1/f noise and its possible role in the transport of the enzymatic components of binary toxins.

INCUBATION TYPE PLANAR PATCH CLAMP AS A NEW POTENTIAL TECHNOLOGY FOR DEVELOPING NEURONAL NETWORK HIGH THROUGHPUT SCREENING DEVICES

Ion-channel current recordings based on an incubation type planar patch clamp were first reported in 2008, using HEK293 cells expressed with TRPV1 channels and capsaicin as ligand molecules. At first the success probability (number of devices which normally worked/total number of devices fabricated) was extremely low (several %). Several years later, we have succeeded in significantly decreasing the base line noise by using a salt-bridge-type Ag/AgCl electrode and successfully demonstrated the application of an incubation type planar patch clamp to ligand gated ion-channel biosensors and light gated ion-channel biosensors using HEK293 cells expressed with ChRWR. Furthermore, a spontaneous ion-channel current from a neuronal network was successfully observed by using a planar patch clamp chip, on which the neuronal network was occasionally formed with a soma of a neuron on a micro through-hole. Although the neuronal network was not controlled, this success shows the high potential of realizing a high throughput screening device on the basis of channel current measurements, which contain the most important information on network conditions.

Microfabrication of PMMA sensor chips for an incubation-type planar patch clamp

A sensor chip for use in an ion channel biosensor based on an incubation-type planar patch clamp was formed using both side hot embossing and focused ion beam (FIB) processing. A biosensor was constructed using the PMMA chip, and the performance of this chip was investigated by measuring the laser-stimulated ion channel current of a channel rhodopsin wide receiver (ChRWR) expressed on HEK293 cells. The observed current profile and membrane potential dependences agreed well with the data measured using a pipette patch clamp. The noise level of the channel current recorded using a 1 kHz low pass filter was 7 pA (rms). The sensor chip fabrication processes are readily modified to permit multi-point measurements; therefore, the excellent performance of the PMMA chip indicates that the chip is suitable for use in high-throughput ion channel biosensor screening applications.

Natural and artificial ion channels for biosensing platforms

The single-molecule selectivity and specificity of the binding process together with the expected intrinsic gain factor obtained when utilizing flow through a channel have attracted the attention of analytical chemists for two decades. Sensitive and selective ion channel biosensors for high-throughput screening are having an increasing impact on modern medical care, drug screening, environmental monitoring, food safety, and biowarefare control. Even virus antigens can be detected by ion channel biosensors. The study of ion channels and other transmembrane proteins is expected to lead to the development of new medications and therapies for a wide range of illnesses. From the first attempts to use membrane proteins as the receptive part of a sensor, ion channels have been engineered as chemical sensors. Several other types of peptidic or nonpeptidic channels have been investigated. Various gating mechanisms have been implemented in their pores. Three technical problems had to be solved to achieve practical biosensors based on ion channels: the fabrication of stable lipid bilayer membranes, the incorporation of a receptor into such a structure, and the marriage of the modified membrane to a transducer. The current status of these three areas of research, together with typical applications of ion-channel biosensors, are discussed in this review.

Poly(aniline) nanowires in sol-gel coated ITO: a pH-responsive substrate for planar supported lipid bilayers.

Facilitated ion transport across an artificial lipid bilayer coupled to a solid substrate is a function common to several types of bioelectronic devices based on supported membranes, including biomimetic fuel cells and ion channel biosensors. Described here is fabrication of a pH-sensitive transducer composed of a porous sol-gel layer derivatized with poly(aniline) (PANI) nanowires grown from an underlying planar indium-tin oxide (ITO) electrode. The upper sol-gel surface is hydrophilic, smooth, and compatible with deposition of a planar supported lipid bilayer (PSLB) formed via vesicle fusion. Conducting tip AFM was used to show that the PANI wires are connected to the ITO, which convert this electrode into a potentiometric pH sensor. The response to changes in the pH of the buffer contacting the PANI nanowire/sol-gel/ITO electrode is blocked by the very low ion permeability of the overlying fluid PSLB. The feasibility of using this assembly to monitor facilitated proton transport across the PSLB was demonstrated by doping the membrane with lipophilic ionophores that respond to a transmembrane pH gradient, which produced an apparent proton permeability several orders of magnitude greater than values measured for undoped lipid bilayers.

Porous Materials to Support Bilayer Lipid Membranes for Ion Channel Biosensors

To identify materials suitable as membrane supports for ion channel biosensors, six filter materials of varying hydrophobicity, tortuosity, and thickness were examined for their ability to support bilayer lipid membranes as determined by electrical impedance spectroscopy. Bilayers supported by hydrophobic materials (PTFE, polycarbonate, nylon, and silanised silver) had optimal resistance (14–19 GΩ) and capacitance (0.8–1.6 μF) values whereas those with low hydrophobicity did not form BLMs (PVDF) or were short-lived (unsilanised silver). The ability of ion channels to function in BLMs was assessed using a method recently reported to improve the efficiency of proteoliposome incorporation into PTFE-supported bilayers. Voltage-gated sodium channel activation by veratridine and inhibition by saxitoxin showed activity for PTFE, nylon, and silanised silver, but not polycarbonate. Bilayers on thicker, more tortuous, and hydrophobic materials produced higher current levels. Bilayers that self-assembled on PTFE filters were the longest lived and produced the most channel activity using this method.

Bilayer lipid membranes supported on Teflon filters: A functional environment for ion channels

Many ion channel proteins have binding sites for toxins and pharmaceutical drugs and therefore have much promise as the sensing entity in high throughput technologies and biosensor devices. Measurement of ionic conductance changes through ion channels requires a robust biological membrane with sufficient longevity for practical applications. The conventional planar BLM is 100–300 μm in diameter and typically contains fewer than a dozen channels whereas pharmaceutical screening methods in cells use current recordings for many ion channels. We present a new, simple method for the fabrication of a disposable porous-supported bilayer lipid membrane (BLM) ion channel biosensor using hydrated Teflon (polytetrafluoroethylene, PTFE) filter material (pore size 5 μm, filter diameter = 1 mm). The lipid layer was monitored for its thickness and mechanical stability by electrical impedance spectroscopy. The results showed membrane capacitances of 1.8 ± 0.2 nF and membrane resistances of 25.9 ± 4.1 GΩ, indicating the formation of lipid bilayers. The current level increased upon addition of the pore-forming peptide gramicidin. Following addition of liposomes containing voltage-gated sodium channels, small macroscopic sodium currents (1–80 pA) could be recorded. By preloading the porous Teflon with sodium channel proteoliposomes, prior to BLM formation, currents of 1–10 nA could be recorded in the presence of the activator veratridine that increased with time, and were inhibited by tetrodotoxin. A lack of rectification suggests that the channels incorporated in both orientations. This work demonstrates that PTFE filters can support BLMs that provide an environment in which ion channels can maintain their functional activity relevant for applications in drug discovery, toxin detection, and odour sensing.

Ion-Channel Biosensors—Part I: Construction, Operation, and Clinical Studies

This paper deals with the construction and operation of a novel biosensor that exploits the molecular switching mechanisms of biological ion channels. The biosensor comprises gramicidin A channels embedded in a synthetic tethered lipid bilayer. It provides a highly sensitive and rapid detection method for a wide variety of analytes. In this paper, we outline the fabrication and principle of operation of the ion-channel switch (ICS) biosensor. The results of a clinical study, in which the ion-channel biosensor is used to detect influenza A in untreated clinical samples, is presented to demonstrate the utility of the technology. Fabrication of biochip arrays using silicon chips decorated with ¿ink jet¿ printing is discussed. We also describe how such biochip arrays can be used for multianalyte sensing. Finally, reproducibility/stability issues of the biosensor are addressed.

Noise Properties of Incubation-Type Planar Ion Channel Biosensor

Current noise and its power spectrum have been measured by an incubation-type planar ion channel biosensor. The dominant noise sources are classified into (i) current noise induced by head stage preamplifier input voltage noise, (ii) thermal noise, and (iii) excess noise. The spectral density and variance of these noises are formulated in the general form using an equivalent circuit. The observed noise in the device has been explained by the excess noise, which depends on 1/ f and originates primarily from the current passing through the cleft between the cell membrane and the substrate surface.

An artificial ion channel biosensor to identify potential anti-HIV drugs

An artificial ion channel biosensor to identify potential anti-HIV drugs

Artificial Ion Channel Biosensor in Human Immunodeficiency Virus gp41 Drug Sensing

An ion channel biosensor is described for label-free detection of inhibitors which bind to the coiled coil domain of human immunodeficiency virus type 1 gp41. gp41 is the viral transmembrane glycoprotein responsible for fusion between HIV-1 and host cells. The N-terminal coiled coil domain binds three antiparallel C-heptad repeat peptides in the six helix bundle structure of trimeric gp41 that forms during fusion. Compounds able to prevent six-helix bundle formation by binding to the gp41 coiled coil could inhibit fusion and have important therapeutic potential. We have immobilized on gold a positively charged metallopeptide mimic of a section of the gp41 coiled coil containing a hydrophobic pocket suitable for small molecule binding. We demonstrate that the resulting sensor is able to transmit a current in the presence of negatively charged redox marker ions, therefore acting as an artificial ion channel. The electrochemical signal, measured by cyclic voltammetry, was modulated by specific analyte binding to the coiled coil. Nanomolar quantities of peptides and small molecules that bind in the hydrophobic pocket could be selectively detected, providing a method for label-free detection of binding to gp41.

Incubation type Si-based planar ion channel biosensor

A new planar-type ion channel biosensor with the function of cell culture has been fabricated using silicon on an insulator substrate as the sensor chip material. Coating of the sensor chip with fibronectin was essentially important for cell incubation on the chip. Although the seal resistance was quite low (approximately 7 Mohms) compared with the pipette patch-clamp gigaohm seal, the whole-cell channel current of the transient receptor potential vanilloid type 1 (TRPV1) channel expressing HEK293 cells was successfully observed, with a good signal-to-noise ratio, using capsaicin as a ligand molecule.

Fabrication of Si-based planar type patch clamp biosensor using silicon on insulator substrate

The aim of this paper is to fabricate the planar type patch clamp ion-channel biosensor, which is suitable for the high throughput screening, using silicon-on-insulator (SOI) substrate. The micropore with 1.2 μm diameter is formed through the top Si layer and the SiO 2 box layer of the SOI substrate by focused ion beam (FIB). Then the substrate is assembled into the microfluidic circuit. The human embryonic kidney 293 (HEK-293) cell transfected with transient receptor potential vanilloid type 1 (TRPV1) is positioned on the micropore and the whole-cell configuration is formed by the suction. Capsaicin is added to the extracellular solution as a ligand molecule, and the channel current showing the desensitization unique to TRPV1 is measured successfully.

Fabrication of a Planar Type Patch-clamp Biosensor using Silicon on Insulator Substrate

We have fabricated a planar type ion channel biosensor useful for the high throughput screening by using SOI (Silicon On Insulator) substrates. The micro-pore of about 1μm diameter was formed through the SOI substrate and controlled the structure of the pore. The substrate was assembled into the micro fluid circuit. Comparing with classical patch-clamp using the pipette, the planar type patch-clamp has advantages not only in the miniaturization and the high integration, but also in easiness of intracellular fluids exchange and of simultaneous measurement of ion channel current and fluoresce imaging. The HEK293 (Human Embryonic Kidney 293) cell with transfected TRPV1 (transient receptor potential vanilloid type 1) channels, which are mainly expressed in sensory nerve cells, was positioned on the micro-pore, and the capsaicin, which is a pungent ingredient of the hot pepper, was added to the external cell solution as a ligand molecule, then the whole-cell current of the ion channel was measured.

S02I5 イオンチャンネルバイオセンサーの開発と応用(分子コンピュータと分子通信:生物機能を利用した新たな情報処理・情報通信システム,シンポジウム,第45回日本生物物理学会年会)

S02I5 イオンチャンネルバイオセンサーの開発と応用(分子コンピュータと分子通信:生物機能を利用した新たな情報処理・情報通信システム,シンポジウム,第45回日本生物物理学会年会)

An anchored PKA and PDE4 complex regulates subplasmalemmal cAMP dynamics

The spatiotemporal regulation of cAMP can generate microdomains just beneath the plasma membrane where cAMP increases are larger and more dynamic than those seen globally. Real-time measurements of cAMP using mutant cyclic nucleotide-gated ion channel biosensors, pharmacological tools and RNA interference (RNAi) were employed to demonstrate a subplasmalemmal cAMP signaling module in living cells. Transient cAMP increases were observed upon stimulation of HEK293 cells with prostaglandin E1. However, pretreatment with selective inhibitors of type 4 phosphodiesterases (PDE4), protein kinase A (PKA) or PKA/A-kinase anchoring protein (AKAP) interaction blocked an immediate return of subplasmalemmal cAMP to basal levels. Knockdown of specific membrane-associated AKAPs using RNAi identified gravin (AKAP250) as the central organizer of the PDE4 complex. Co-immunoprecipitation confirmed that gravin maintains a signaling complex that includes PKA and PDE4D. We propose that gravin-associated PDE4D isoforms provide a means to rapidly terminate subplasmalemmal cAMP signals with concomitant effects on localized ion channels or enzyme activities.

S1e2-6 Fabrication of Ion-channel Biosensor and Interface Control(S1-e2: "New Biomembrane Model Systems, Giant Liposomes and Supported Planar Bilayers, for Probing Biomembrane Structure and Function, and Creation of De Novo Functional Membrane System",Symposia,Abstract,Meeting Program of EABS & BSJ 2006)

S1e2-6 Fabrication of Ion-channel Biosensor and Interface Control(S1-e2: "New Biomembrane Model Systems, Giant Liposomes and Supported Planar Bilayers, for Probing Biomembrane Structure and Function, and Creation of De Novo Functional Membrane System",Symposia,Abstract,Meeting Program of EABS & BSJ 2006)

Formation of high-resistance supported lipid bilayer on the surface of a silicon substrate with microelectrodes

We have developed two basic technologies for fabrication of supported planar lipid bilayer membrane ion channel biosensors: a defect-free lipid bilayer formation on the substrate surface with electrode pores and a patterning technique for the hydrophobic self-assembled-monolayer to form the guard ring that reduces the lipid bilayer edge-leak current. The importance of the supported-membrane structure to achieve low noise and high-speed performance is suggested on the basis of the observed relation between the single-ion-channel current noise and the pore size.

Electrochemical biosensor based on supported planar lipid bilayers for fast detection of pathogenic bacteria

This paper presents a new ion-channel biosensor based on supported bilayer lipid membrane for direct and fast detection of Campylobacter species. The sensing element of a biosensor is composed of a stainless-steel working electrode, which is covered by artificial bilayer lipid membrane (BLM). Antibodies to bacteria embedded into the BLM are used as channel forming proteins. The biosensor has a strong signal amplification effect, which is defined as the total number of ions transported across the BLM. The total number of (univalent) ions flowing through the channels is 10 10 ions s −1. The biosensor showed a very good sensitivity and selectivity to Campylobacter species.