Transport across artificial membranes–an analytical perspective

Biosensors that make use of transport processes across lipid membranes are very rare even though a stimulus, the binding of a single analyte molecule, can enhance the sensor response manifold if the analyte leads to the transport of more than one ion or molecule across the membrane. Prerequisite for a proper function of such membrane based biosensors is the formation of lipid bilayers attached to a support that allow for the insertion of membrane peptides and proteins in a functional manner. In this review, the current state of the art technologies to obtain lipid membranes on various supports are described. Solid supported membranes on transparent and electrically conducting surfaces, lipid bilayers on micromachined apertures and on porous materials are discussed. The focus lies on the applicability of such membranes for the investigation of transport phenomena across lipid bilayers facilitated by membrane embedded peptides, channel proteins and transporters. Carriers and channel forming peptides, which are easy to handle and rather robust, are used frequently to build up membrane based biosensors. However, channel forming proteins and transporters are more difficult to insert functionally and thus, there are yet only few examples that demonstrate the applicability of such systems as biosensor devices.      

Tripodal, cooperative, and allosteric transphosphorylation metallocatalysts

Three artificial amino acids derived from l-serine by replacing the hydroxyl moiety with 1,4,7-triazacyclononane, 1,5,9-triazacyclododecane, and 1,4,7,10-tetraazacyclododecane, respectively, have been connected to the three arms of the tetraamine tris(2-aminoethyl)amine, Tren, to obtain tripodal ligands. They are able to bind up to four metal ions (like CuII and ZnII), three with the polyazamacrocycles and one with the Tren platform. Some of the ZnII complexes of these tripodal ligands proved to be good catalysts for the cleavage of the RNA model substrate 2-hydroxypropyl-p-nitrophenylphosphate (HPNP). Studies of the catalytic activity in the presence of increasing amounts of ZnII show that the complexes represent minimalist examples of metallocatalysts with cooperativity between the metal centers and allosteric control by a metal ion. The Tren binding site constitutes the allosteric regulation unit, while the three ZnII-azacrown complexes provide the cooperative, catalytic site. The allosteric role of the ZnII ion located in the Tren binding site was unambiguously demonstrated by studying the catalytic activity of a derivative unable to complex ZnII in that site. In this case, the cooperativity between the three ZnII ions bound to the peripheral azacrowns was totally suppressed. The kinetic analysis has shown that cooperativity is due to neither the occurrence of general-acid/general-base catalysis nor a decreased binding of the substrate because of the deprotonation of a water molecule bound to the complex but, rather, stabilization of the complexed substrate in its transformation into the transition state.     

Conditioning saliva for use in a microfluidic biosensor

This report details an approach to saliva conditioning for compatibility of raw patient samples with microfluidic immunoassay components, principally biosensor surfaces susceptible to fouling. Stimulated whole human saliva spiked with a small molecule analyte (phenytoin, 252 Da) was first depleted of cells, debris and high molecular weight glycoproteins (mucins) using membrane filtration. This process significantly reduced but did not eliminate fouling of biosensor surfaces exposed to the sample. An H-filter, which separates solutes from mixed samples based on their diffusion in laminar flow, was used to extract the analyte from the remaining large molecular weight species in the filtered saliva sample. Patient samples treated in this way retained 23% of the analyte with 97% and 92% reduction in glycoproteins and proteins, respectively, and resulted in 3.6 times less surface fouling than either untreated or filtered saliva alone. These sample conditioning steps will enable the use of fouling-sensitive detection techniques in future studies using clinical saliva samples.     

Dielectrophoretic cell trapping for improved surface plasmon resonance imaging sensing

The performance of conventional surface plasmon resonance (SPR) biosensors can be limited by the diffusion of the target analyte to the sensor surface. This work presents an SPR biosensor that incorporates an active mass‐transport mechanism based on dielectrophoresis and electroosmotic flow to enhance analyte transport to the sensor surface and reduce the time required for detection. Both these phenomena rely on the generation of AC electric fields that can be tailored by shaping the electrodes that also serve as the SPR sensing areas. Numerical simulations of electric field distribution and microparticle trajectories were performed to choose an optimal electrode design. The proposed design improves on previous work combining SPR with DEP by using face‐to‐face electrodes, rather than a planar interdigitated design. Two different top‐bottom electrode designs were experimentally tested to concentrate firstly latex beads and secondly biological cells onto the SPR sensing area. SPR measurements were then performed by varying the target concentrations. The electrohydrodynamic flow enabled efficient concentration of small objects (3 μm beads, yeasts) onto the SPR sensing area, which resulted in an order of magnitude increased SPR response. Negative dielectrophoresis was also used to concentrate HEK293 cells onto the metal electrodes surrounded by insulating areas, where the SPR response was improved by one order of magnitude.     

Biospecific interaction analysis using surface plasmon resonance detection applied to kinetic, binding site and concentration analysis.

A system for real-time biospecific interaction analysis using biosensor technology based on the optical phenomenon surface plasmon resonance is described. The biospecific interface is a sensor chip covered with a hydrogel matrix. One component of the interaction to be studied is immobilized covalently to the hydrogel and other interactants are passed over the chip in solution. The mass change at the sensor surface, reflecting the progress of the interaction studied, is monitored in real time. The technique, which does not require molecular labels for detection, can measure mass changes down to 10 pg/mm2. Repeated analyses can be performed on the same sensor chip. Applications shown include kinetic measurements, binding site analysis and concentration determination.     

Dynamics of Pseudomonas aeruginosa association with anionic hydrogel surfaces in the presence of aqueous divalent-cation salts

Binding of bacteria to solid surfaces is complex with many aspects incompletely understood. We investigate Pseudomonas aeruginosa uptake kinetics onto hydrogel surfaces representative of soft-contact lenses made of nonionic poly(2-hydroxyethylmethacrylate) (p-HEMA), anionic poly(methacrylic acid) (p-MAA), and anionic poly(acrylic acid) (p-AA). Using a parallel-plate flow cell under phase-contrast microscopy, we document a kinetic "burst" at the anionic hydrogel surface: dilute aqueous P. aeruginosa first rapidly accumulates and then rapidly depletes. Upon continuing flow, divalent cations in the suspending solution sorb into the hydrogel network causing the previously surface-accumulated bacteria to desorb. The number of bacteria eventually bound to the surface is low compared to the nonionic p-HEMA hydrogel. We propose that the kinetic burst is due to reversible divalent-cation bridging between the anionic bacteria and the negatively charged hydrogel surface. The number of surface bridging sites diminishes as divalent cations impregnate into and collapse the gel. P. aeruginosa association with the surface then falls. Low eventual binding of P. aeruginosa to the anionic hydrogel is ascribed to increased surface hydrophilicity compared to the counterpart nonionic p-HEMA hydrogel.     

Mechanisms controlling the sensitivity of amperometric biosensors in flow injection analysis systems

This paper numerically investigates the sensitivity of an amperometric biosensor acting in the flow injection mode when the biosensor contacts an analyte for a short time. The analytical system is modelled by non-stationary reaction-diffusion equations containing a non-linear term related to the Michaelis-Menten kinetics of an enzymatic reaction. The mathematical model involves three regions: the enzyme layer where enzymatic reaction as well as the mass transport by diffusion takes place, a diffusion limiting region where only the diffusion takes place, and a convective region. The biosensor operation is analysed with a special emphasis to the conditions at which the biosensor sensitivity can be increased and the calibration curve can be prolonged by changing the injection duration, the permeability of the external diffusion layer, the thickness of the enzyme layer and the catalytic activity of the enzyme. The apparent Michaelis constant is used as a main characteristic of the sensitivity and the calibration curve of the biosensor. The numerical simulation was carried out using the finite difference technique.     

Cooperative properties of hormone receptors in cell membranes.

The binding of many polypeptide hormones to cell surface receptors does not appear to follow the law of mass action. While steady-state binding data are consistent in many cases with either heterogeneous populations of binding sites or interactions of the type known as negative cooperativity, study of the kinetics of dissociation of the type known as negative cooperativity, study of the kinetics of dissociation of the hormone receptor complex allows an unambiguous demonstration of cooperative interactions. Negative cooperativity, which seems to be wide-spread among hormone receptors, provides exquisite sensitivity of the cell at low hormone concentrations while buffering against acutely elevated hormone levels. The molecular mechanisms underlying the cooperativity are still largely unknown. Cooperativity may stem from a conformational transition in individual receptors or involve receptor aggregation in the fluid membrane (clustering) or more extensive membrane phenomena. Thus, new models of hormone action must be considered which integrate the progress in our knowledge of both the complex mechanisms regulating hormone binding to their surface receptors, and the dynamic properties of the cell membrane.     

Free energy calculations on the two drug binding sites in the M2 proton channel

Two alternative binding sites of adamantane-type drugs in the influenza A M2 channel have been suggested, one with the drug binding inside the channel pore and the other with four drug molecule S-binding to the C-terminal surface of the transmembrane domain. Recent computational and experimental studies have suggested that the pore binding site is more energetically favorable but the external surface binding site may also exist. Nonetheless, which drug binding site leads to channel inhibition in vivo and how drug-resistant mutations affect these sites are not completely understood. We applied molecular dynamics simulations and potential of mean force calculations to examine the structures and the free energies associated with these putative drug binding sites in an M2-lipid bilayer system. We found that, at biological pH (~7.4), the pore binding site is more thermodynamically favorable than the surface binding site by ~7 kcal/mol and, hence, would lead to more stable drug binding and channel inhibition. This result is in excellent agreement with several recent studies. More importantly, a novel finding of ours is that binding to the channel pore requires overcoming a much higher energy barrier of ~10 kcal/mol than binding to the C-terminal channel surface, indicating that the latter site is more kinetically favorable. Our study is the first computational work that provides both kinetic and thermodynamic energy information on these drug binding sites. Our results provide a theoretical framework to interpret and reconcile existing and often conflicting results regarding these two binding sites, thus helping to expand our understanding of M2-drug binding, and may help guide the design and screening of novel drugs to combat the virus.     

Electrothermal stirring for heterogeneous immunoassays

A technique is proposed to enhance microfluidic immuno-sensors, for example, immunoassays, in which a ligand immobilized on a microchannel wall specifically binds analyte flowing through the channel. These sensors can be limited in both response time and sensitivity by the diffusion of analyte to the sensing surface. In certain applications, the sensitivity and response of these heterogeneous immunoassays may be improved by using AC electrokinetically-driven microscale fluid motion to enhance antigen motion towards immobilized ligands. Specifically, the electrothermal effect is used to micro-stir analyte near the binding surface. Numerical simulations of antigen in a microchannel flow subjected to the electrothermal effect show that 6 V(rms) applied to electrodes near a binding region can increase binding in the first few minutes by a factor of seven. The effectiveness of electrothermal stirring is a strong function of the Damk?hler number. The greatest binding enhancement is possible for high Damk?hler numbers, where the reaction is limited by diffusion. Based on these results, the utility of this technique for diffusion-limited microfluidic sensor applications is demonstrated.     

The Effect of Diffusion Limitations on the Response of Amperometric Biosensors with Substrate Cyclic Conversion

A mathematical model of amperometric biosensors in which chemical amplification by cyclic substrate conversion takes place in a single enzyme membrane has been developed. The model involves three regions: the enzyme layer where enzyme reaction as well as mass transport by diffusion takes place, a diffusion limiting region where only the diffusion takes place, and a convective region where the analyte concentration is maintained constant. Using computer simulation the influence of the thicknesses of the enzyme layer and the diffusion region on the biosensor response was investigated. This paper deals with conditions when the mass transport in the exterior region may be neglected to simulate the biosensor response in a well-stirred solution. The digital simulation was carried out using the finite difference technique.     

Characterisation of morphology of self-assembled PEG monolayers: a comparison of mixed and pure coatings optimised for biosensor applications

For detection of low concentrations of analytes in complex biological matrices using optical biosensors, a high surface loading with capture molecules and a low nonspecific binding of nonrelevant matrix molecules are essential. To tailor biosensor surfaces in such a manner, poly(ethylene glycols) (PEG) in varying lengths were immobilised covalently onto glass-type surfaces in different mixing ratios and concentrations, and were subsequently modified with three different kinds of receptors. The nonspecific binding of a model protein (ovalbumin, OVA) and the maximum loading of the respective analytes to these prepared surfaces were monitored using label-free and time-resolved reflectometric interference spectroscopy (RIfS). The three different analytes used varied in size: 150 kDa for the anti-atrazine antibody, 60 kDa for streptavidin and 5 kDa for the 15-bp oligonucleotide. We investigated if the mixing of PEG in different lengths could increase the surface loadings of analyte mimicking a three-dimensional matrix as was found using dextrans as sensor coatings. In addition, the effect on the surface loading was investigated with regard to the size of the analyte molecule using such mixed PEGs on the sensor surface. For further characterisation of the surface coatings, polarisation modulation infrared reflection absorption spectroscopy, atomic force microscopy, and ellipsometry were applied. All authors contributed equally to this work.     

Analyte response in laser ablation inductively coupled plasma mass spectrometry

The dependence of analyte sensitivity and vaporization efficiency on the operating parameters of an inductively coupled plasma mass spectrometer (ICPMS) was investigated for a wide range of elements in aerosols, produced by laser ablation of silicate glass. The ion signals were recorded for different carrier gas flow rates at different plasma power for two different laser ablation systems and carrier gases. Differences in atomization efficiency and analyte sensitivity are significant for the two gases and the particle size distribution of the aerosol. Vaporization of the aerosol is enhanced when helium is used, which is attributed to a better energy-transfer from the plasma to the central channel of the ICP and a higher diffusion rate of the vaporized material. This minimizes elemental fractionation caused by sequential evaporation and reduces diffusion losses in the ICP. The sensitivity change with carrier gas flow variation is dependent on m/z of the analyte ion and the chemical properties of the element. Elements with high vaporization temperatures reach a maximum at lower gas flow rates than easily vaporized elements. The sensitivity change is furthermore dependent on m/z of the analyte ion, due to the mass dependence of the ion kinetic energies. The mass response curve of the ICPMS is thus not only a result of space charge effects in the ion optics but is also affected by radial diffusion of analyte ions and the mismatch between their kinetic energy after expansion in the vacuum interface and the ion optic settings.     

Modeling of nucleic acid adsorption on 3D prisms in microchannels

Understanding nucleic acid adsorption in microchannels is critical to improve the efficiency of purifying and extracting nucleic acid (NA) from sample solutions by microfluidic technologies. Using a microchannel with 3D prismatic silica elements on the wall can dramatically increase the surface area-to-volume ratio, and hence facilitate the nucleic acid adsorption on the wall. In this study a theoretical model for modeling adsorption in a microchannel with a designed 3D surface structure was developed, and five dimensionless numbers were found to be the key parameters in the adsorption process. Extensive numerical simulations were conducted. Two flow modes, the electroosmotic flow (EOF) and pressure-driven flow (PDF), were investigated for their effect on the adsorption. It was found that the EOF is more desirable than PDF. The 3D prismatic elements can increases the NA molecule adsorption not only by providing more surface areas, but also by the induced pressure resisting the central bulk electroosmotic flow. Finally, the effects of adsorption kinetic parameters (i.e., the kinetic association/dissociation constants, the diffusion coefficient, the total site density, the loading concentration, and the channel height), on the adsorption process were discussed in detail.     

Kinetic discrimination of sequence-specific DNA-drug binding measured by surface plasmon resonance imaging and comparison to solution-phase measurements

We demonstrate the use of surface plasmon resonance (SPR) imaging for direct detection of small-molecule binding to surface-bound DNA probes. Using a carefully designed array surface, we quantitatively discriminate between the interactions of a model drug with different immobilized DNA binding sites. Specifically, we measure the association and dissociation intercalation rates of actinomycin-D (ACTD) to and from double-stranded 5'-TGCT-3' and 5'-GGCA-3' binding sites. The rates measured provide mechanistic information about the DNA-ACTD interaction; ACTD initially binds nonspecifically to DNA but exerts its activity by dissociating slowly from strong affinity sites. We observe a slow dissociation time of kd-1 = 3300 +/- 100 s for ACTD bound to the strong affinity site 5'-TGCT-3' and a much faster dissociation time (210 +/- 15 s) for ACTD bound weakly to the site 5'-GGCA-3'. These dissociation rates, which differ by an order of magnitude, determine the binding affinity for each site (8.8 x 10(6) and 1.0 x 10(6) M(-1), respectively). We assess the effect the surface environment has on these biosensor measurements by determining kinetic and thermodynamic constants for the same DNA-ACTD interactions in solution. The surface suppresses binding affinities approximately 4-fold for both binding sites. This suppression suggests a barrier to DNA-drug association; ACTD binding to duplex DNA is approximately 100 times slower on the surface than in solution.     

Numerical Simulation and Experimental Validation of the Three-Dimensional Flow Field and Relative Analyte Concentration Distribution in an Atmospheric Pressure Ion Source

In this study, the validation and analysis of steady state numerical simulations of the gas flows within a multi-purpose ion source (MPIS) are presented. The experimental results were obtained with particle image velocimetry (PIV) measurements in a non-scaled MPIS. Two-dimensional time-averaged velocity and turbulent kinetic energy distributions are presented for two dry gas volume flow rates. The numerical results of the validation simulations are in very good agreement with the experimental data. All significant flow features have been correctly predicted within the accuracy of the experiments. For technical reasons, the experiments were conducted at room temperature. Thus, numerical simulations of ionization conditions at two operating points of the MPIS are also presented. It is clearly shown that the dry gas volume flow rate has the most significant impact on the overall flow pattern within the APLI source; far less critical is the (larger) nebulization gas flow. In addition to the approximate solution of Reynolds-Averaged Navier-Stokes equations, a transport equation for the relative analyte concentration has been solved. The results yield information on the three-dimensional analyte distribution within the source. It becomes evident that for ion transport into the MS ion transfer capillary, electromagnetic forces are at least as important as fluid dynamic forces. However, only the fluid dynamics determines the three-dimensional distribution of analyte gas. Thus, local flow phenomena in close proximity to the spray shield are strongly impacting on the ionization efficiency.     

Coupling a natural receptor protein with an artificial receptor to afford a semisynthetic fluorescent biosensor

An artificial receptor and a signal transducer have been engineered on a lectin (saccharide-binding protein) surface by a post-photoaffinity labeling modification method. Saccharide binding can be directly and selectively read out by the fluorescence changes of the fluorophore via photoinduced electron transfer (PET) mode. Fluorescence titration with various saccharides reveals that molecular recognition by the artificial receptor is successfully coupled to the native binding site of the lectin, producing a novel fluorescent saccharide biosensor showing modulated specificity and enhanced affinity. Designed cooperativity between artificial and native molecular recognition modules was quantitatively demonstrated by the comparison of the binding affinities, and it represents a new strategy in molecular recognition. By using appropriate artificial receptors and various native lectins, this approach may provide many new semisynthetic biosensors for saccharide derivatives such as glycolipids and glycopeptides/proteins. An extended library of lectin-based biosensors is envisioned to be useful for glycome research, a newly emerging field of the post-genomic era.     

Evaluation of electrodeposited gold nanostructures for applications in QCM sensing

The electrodeposition of gold nanostructures increases the surface area of a biosensor, which brings an enhancement of the sensitivity by increasing the amount of analyte binding to the surface. To evaluate the relationship among the surface structure, the area and the analyte binding, we quantitatively analyzed them for quartz crystal microbalance (QCM) sensing by scanning electron microscopy and cyclic voltammetry measurements. The results indicate a several-times increase of analyte bindings, and also the limitation of the sensing performance.     

A Fractal Analysis Approach for the Evaluation of Hybridization Kinetics in Biosensors

The diffusion-limited hybridization kinetics of analyte in solution to a receptor immobilized on a biosensor or immunosensor surface is analyzed within a fractal framework. The data may be analyzed by a single- or a dual-fractal analysis. This was indicated by the regression analysis provided by Sigmaplot (Sigmaplot, Scientific Graphing Software, User's Manual, Jandel Scientific, CA, 1993). It is of interest to note that the binding rate coefficient and the fractal dimension both exhibit changes, in general, in the same direction for both the single-fractal and the dual-fractal analysis examples presented. The binding rate coefficient expression developed as a function of the analyte concentration in solution and the fractal dimension is of particular value since it provides a means to better control biosensor or immunosensor performance. Copyright 2001 Academic Press.     

Theoretical Assessment of Binding and Mass‐Transport Effects in Electrochemical Affinity Biosensors That Utilize Nanoparticle Labels for Signal Amplification

This paper presents a theoretical study of electrochemical affinity biosensors for the detection of DNA/protein that utilize nanoparticle labels for signal amplification. This study analyzes the effects of binding and mass transport of the analytes on biosensor performance by using numerical simulations. Four cases were considered: 1) nanoparticles used to increase the loading of an electroactive species, or used as catalysts under pseudo‐first‐order conditions; 2) nanoparticles used as ultramicroelectrode arrays for the electrolysis of large concentrations of substrate; 3) nanoparticles used as seeds to deposit electrochemically detectable species; and 4) nanoparticles used to mediate the deposition of electrocatalysts. By using nanoparticle labels, high sensitivity is possible under all conditions considered. However, theoretical findings suggested that nonspecific adsorption could be more problematic in cases 2–4 due to the mismatch between the chemistry of surface binding and the principle of signal amplification that originates from the effect of mass transport. Under these conditions, any given signal would plateau at a much lower analyte concentration, well before the analyte binding had actually reached a plateau. Views on possible solutions to the above limitations are also presented.