3 CHAPTER Nanoporous membrane for the selective transport of charged proteins 3.1 INTRODUCTION 3.1.1 Biomimetic membrane systems In 1969, Otto H Schmitt coined the term ‘biomimetic’ The term, itself is derived from ‘bios’, meaning life and ‘mimesis’, meaning to imitate Thus biomimetic work represents the study and imitation of natural methods, designs and processes While some processes or designs are copied, many ideas from nature are best adapted when they serves as inspiration for human-made capabilities [1] Development of biomimetic systems to mimic cell membrane structures play important role in investigating biological cellular processes [2, 3] and has wide applications ranging from biosensors and therapy in medicine and pharmaceutical science to artificial photosynthetic systems in green chemistry 65 Several studies in the past thirty years have focused on constructing an artificial bilayer lipid membrane that can act as cell membranes [4, 5] The first artificial lipid membrane system was developed by Mueller et al in 1960s [6], and was termed as black lipid membrane, which is the planar phospholipids layers system, consisted of bilayer lipid membranes (BLMs) structure located between two compartments containing aqueous solutions The BLMs structure functions as a barrier that prevents transportation of ions and other charged species Tadini et al introduced the P-types ATPase which is an integral membrane protein into the BLMs to study ion transport in the ATPase [7] Other types of ATPase (Na+-K+ or Ca2+) were also embedded in the BLMs to achieve active transport of selective ions through the membranes The black lipid membranes are suspended freely in the solution, therefore they are mobile and active However, this limits the lifetime of the bilayer due to the poor stability of the membrane Moreover, the analytical techniques used to study BLMs system are limited Subsequently, more stable membranes were developed using solid substrates to support the lipid membranes, which are known as “supported lipid bilayers” A large number of supported lipid bilayer systems including 66 solid-supported lipid bilayers, polymer-cushioned lipid bilayers, hybrid bilayers, tethered lipid bilayers, suspended lipid bilayers, and supported vesicular layers have been studied Naumann et al investigated the cation selectivity of valinomycin in tethered lipid bilayers on gold surfaces The impedance spectroscopy was employed to control the specific transport of potassium ion through the tethered lipid bilayer system [8] Nikolelis et al detected ammonium ion by stabilizing the gramicidin in the metal supported bilayer membrane [9] They also used self-assembled lipid membrane and the transport of DNA through the membrane to detect DNA-hybridization [10] Since supported lipid bilayers are tagged on the substrate surface, the mobility and activity of the membranes are somewhat restricted; hence the stability of the membrane improves However, this restriction of activity presents some disadvantages to supported lipid bilayer in cases where the mobility of protein enzyme constituted in the membranes is necessary Besides, the preparation of supported lipid bilayer membranes requires specialized skills and knowledge Besides the studies on artificial lipid membranes, other synthetic membrane structures with or without incorporation of chemical or biological functional groups were used to mimic the membrane function of selective 67 transport For example, Bernard’s group prepared the composite enzymatic membrane (CEM) to separate the D, L forms of a racemic mixture of (D/L) [11], and to isolate and to concentrate the L form Martin et al could achieve fivefold difference between the transport rates of D- and L-amino acids through a microporous polypyrrole/polycarbonate/polypyrrole sandwich membrane immobilized with apoenzymes within the membrane channel [12] Shufang et al used gold nanotube membrane derivatized with poly(ethylene glycol) to separate some proteins such as lysozyme, bovine serum albumin and β-lactoglobulin A [13] Instead of using biological or chemical function groups, other studies exploited the external charged potential to drive the transport of the analytes across the synthetic membranes Recent works by White et al demonstrated the use of the electrostatic and photochemical controls for specific transport of different charged species through the ion-exchange membrane [14, 15] In their later work, Matin’s and Strove’s groups also employed transmembrane potentials to produce driving forces for both flux and electrophoresis selectivity of proteins across the gold-coated nanotube transmembrane based on their differences on protein charges [16-18] Yamauchi et al investigated the diffusion of charged trypsin through the 68 polyelectrolyte PVA/PAA membrane by an externally applied electric field [19] In many of previous studies, the electric field was applied far across the membrane using inert electrodes immersed in the cell compartments on either side of the membrane This reduced the magnitude of applied potential across the membrane due to ohmic resistance in the bulk solution, causing potential drop especially when current passed was high The nanoporous alumina membranes, a type of ceramic membranes, have been used to study the transport of analytes through membranes One advantage of alumina membrane is its controllable pore size The pore size of alumina membrane can be adjusted from few nm to few hundreds of nm depending on the fabrication methods In the early work, Bluhm et al studied transport behavior of monovalent and divalent solutes across mesoporous Anopore γ-alumina membranes as a function of pore diameter, pH, ionic strength, and nature of the salt or complexing species in solution [20] They proposed a correlation between cation selectivity and membrane structure, which has become the basis for later work with alumina membranes In another work, Teramae et al studied the diffusion of metal complexes and charged organic dye molecules through the silica-surfactant nanochannels in 69 porous alumina membrane [21, 22] In our work, the nanoporous alumina membrane was employed to study the transports of different charged proteins through the membrane channels We focused on the influence of transmembrane potentials on the transport behavior of differently charged proteins The alumina membrane was fabricated in same way as the electrode-membrane-electrode system used for the collection and shielding experiments of ferrocenes, in Chapter Unlike other works [19] in which the biological analytes were transported through the membranes by an external electrical field applied at electrodes placed some distances away from the membranes, in our study the electric field was applied directly at the platinum-coated layers of a micrometer-thick membrane This has the advantage of achieving high field strengths of about 30 kV m−1 suitable for electrokinetic transport of proteins using very small transmembrane potential of V Three common proteins with different charges were used in this work They were positively charged lysozyme, negatively charged bovine serum albumin and neutral haemoglobin in buffer solutions of pH 70 3.1.2 Lysozyme Lysozyme from chicken egg was first described by Laschtschenko in 1909 Lysozyme (Lys) is widely distributed and is found in not only egg white but also in many animal tissue and secretions, and some fungi Alternative names for lysozyme N,O-diacetylmuramidase, Lysozyme g, are 1,4-N-acetylmuramidase, PR1-Lysozyme, Mucopeptide Globulin G1, N-acetylmuramoylhydrolase, L-7001, Globulin G, Mucopeptide glucohydrolase and Muramidase Lys is a peptide chain which contains 129 amino acids and four disulfide bonds, with a molecular weight of 14.3 kDa, and the theoretical isoelectric point of 11 Lys has wide range of applications, such as in nucleic acid or plasmid preparation, chitin/bacterial cell walls hydrolysis, or protein purification from inclusion bodies [23] 3.1.3 Bovine serum albumin Bovine serum albumin (BSA) which is also known as “Fraction V” is a large globular protein (66000 Dalton, dimension of 40×40×140 Å3) [24] The name “Fraction V” refers to the albumin in the fifth fraction of the original Edwin Cohn purification method By changing solvent concentrations, pH, salt levels, and temperature, Edwin Cohn was able to pull out successive 71 "fractions" of blood plasma Bovine serum albumin (BSA) makes up approximately 60% of all proteins in animal serum BSA is stable, and not affected in many biochemical reactions Moreover, BSA is easily purified in large amount from bovine blood, a by-product of cattle industry; therefore, it has wide applications BSA is used as the standard protein in some methods to quantify other proteins BSA can stabilize some enzymes during digestion of DNA and prevent the adhesion of enzymes to reaction tube and vessels BSA protein is composed of 583 amino acid residues with overall isoelectronic point of 4.7 in water at 250C 3.1.4 Myoglobin Myoglobin (Mb) is a single-chain globular protein which contains 153 amino acids It has a molecular weight of 17800 Daltons Isoelectric point in water at 25oC of Mb from horse is 6.9 Mb is found in cardiac and red skeletal muscles, where it functions in the storage of oxygen and facilitates oxygen transport to the mitochondria for oxidative phosphorylation Mb is abundant in diving mammals such as whales and seals, helping them to hold breath longer 72 3.2 EXPERIMENTAL 3.2.1 Reagents and materials Bovine serum albumin (BSA), lysozyme from chicken egg white (Lys) and myoglobin from horse heart (Mb) were purchased from Sigma-Aldrich All protein solutions were prepared in ultrapure water (Nanopure Ultrapure Water System) in order to increase the Debye length of the charged protein molecules and hence their interaction with the charges on the electrode layer adjacent to the receiver solution The feed concentrations of BSA, Lys and Mb were g L−1, g L−1 and g L−1, and their solution pHs were 7.5, 5.5 and 7.4, respectively 13 mm diameter membrane with 60 μm thickness and 100 nm nominal pore size and membrane holder were purchased from Whatman (Maidstone, Kent, UK) 3.2.2 Preparation of platinum-coated alumina membrane All membranes were washed and pre-treated with 35% hydrogen peroxide (Scharlau) and subsequently sputtered with platinum (99.99% purity) using a JEOL AutoFine Coater (JFC-1600) Sputtering conditions were optimized to achieve sufficiently high conductivity and to maintain the porous structure The thickness of the sputtered platinum layer was about 50 nm A 73 mm thick ring along the outer edge of the membrane was left uncoated to avoid short circuiting when the potential was applied on the two sides of membrane The membrane was placed in a membrane holder made conductive by sputter-coating micrometer thick platinum layers along selective areas to maximize electrically conductive contacts with the membrane The platinum coated regions of the holder were connected to a potentiostat (eDaq EA161) via copper wires The working electrode was connected to the receiver side of the membrane while the auxiliary and reference leads were attached to the feed side of the membrane The membrane was left in contact with the feed solution for about before applying the electrical potential Epoxy glue was applied generously to insulate and keep all electrical components and connections intact 3.3 RESULT AND DISCUSSION 3.3.1 Fluxes of single proteins under the influence of electrical potential Fig 3.1 shows the schematics of the platinum-coated alumina membrane system All protein solutions prepared from the stock solution were stored at o C and used within three days of preparation mL protein solution was 74 contained conductive gold throughout the nanopore walls In addition, the interaction of proteins with the platinum layer of the platinum-coated alumina membrane was expected to be less significant, due to the large pore dimension of the platinum layer (ca 60 nm) in comparison with the protein size, described elsewhere [18].Conversely, BSA flux increased as the transmembrane potential increased in the positive direction The isoelectric point of BSA at 4.7 [23, 25, 26] was lower than the solution pH of 7.5, thus the protein was negatively charged At zero potential, the BSA flux was 1.5 μmol m-2 s-1, similar to that found for the transport rate of neutral BSA across a track etched polycarbonate membrane modified by charged self-assembled monolayers at pH 4.7 [27] The neutral BSA molecules have minimal interactions with the membrane surfaces and thus, its transport was not influenced by the presence of surfaces along the membrane walls At unfavourable potentials negative of zero potential, the BSA flux remained fairly constant This was similarly observed for Lys fluxes under unfavourable potentials On the other hand, the Mb flux was little affected by the applied potentials, as expected since the isoelectric point of Mb was 6.9 [26], very close to Mb solution pH of 6.7 78 3.3.2 Fluxes of proteins in a mixed protein experiment In the mixed protein experiment, all the proteins absorbed at 280 nm Thus it was necessary to correct for the interfering absorbance intensities by BSA and Mb, in order to derive the concentration of Lys from the 280 nm peak intensity Concentrations of the colored BSA and Mb were derived from -2 -1 Flux(μ mol m s ) the 610 nm and 410 nm absorbance peaks directly without further corrections 25 20 15 10 BSA LYS Mb -2 -1 Potential (V) Fig 3.3 Effect of transmembrane potentials on the initial fluxes of BSA, Lys and Mb in a mixed protein experiment Generally, the protein fluxes followed closely the trend observed in single protein experiments under different applied potentials between -1.5 V and +1.5 V Fig 3.3 shows the large variation in the flux ratio of Lys:BSA:Mb from 96:1:12 at -1.5 V to 5:1:50 at zero potential to 2:2:1 at +1.5 V However, 79 the protein fluxes in both single and mixed experiments differed significantly for Lys and BSA at zero potential At zero potential, Lys and BSA fluxes were 7.5 and 1.4 μmol m-2 s-1 respectively in the single protein experiments, but decreased to ca 0.3 and 0.1 μmol m-2 s-1 in mixed protein experiments In contrast, the change of Mb fluxes from 1.2 to 3.6 μmol m-2 s-1 was less significant At zero transmembrane potential, the protein flux arises from diffusion down the concentration gradient between the feed and receiver solutions ( δC ) with a protein diffusion coefficient (D), according to Ficks’ δx diffusion eqn ( J = D δC ) Since the membrane was pre-equilibrated in the δx feed solution for before the start of the experiment, the feed solution was expected to fill the entire channel, separated from the receiver solution by the 50 nm thick platinum electrode layer Assuming the diffusion layer thickness (δx) was equivalent to the thickness of the platinum electrode layer and using known diffusion coefficients of proteins (DBSA = 6.1×10-11 m2 s-1, DLys = 12.3×10-11 m2 s-1, DMb = 10.2×10-11 m2 s-1) [28], we could estimate the concentrations of proteins accumulated at the electrode/channel solution interface and the diffusion layer thicknesses from the initial protein fluxes For the 80 single protein experiments, the protein concentrations at the electrode/channel solution interface were estimated at 1.6, 2.2 and 1.0 % of feed concentrations and diffusion layer thicknesses of 3.1, 2.2 and 6.0 μm at initial times This concentration polarization however, could not explain why the fluxes for Lys and BSA were significantly lower in the mixed protein experiment For the mixed protein experiment, the diffusion layer thickness calculated from the initial Lys and BSA fluxes at zero transmembrane potential was found to be ca 50 μm, similar to the actual membrane thickness This strongly suggested some forms of interaction between Lys and BSA which significantly decreased the amount of these two proteins in the channels during the pre-equilibration time These interactions could arise from ion-pairing between the oppositely charged Lys and BSA molecules [27] which subsequently formed protein clusters (dimers or trimers) and aggregated along the channel walls The larger protein clusters were expected to move slower than the single protein molecules through the nanopore [17, 27] The ion-pairing effect however, would not affect the somewhat neutral Mb, which reasonably explained the relatively unchanged Mb flux in the mixed protein experiment It was also possible the differences in the Lys and BSA fluxes between the single and mixed protein experiments at zero transmembrane 81 potential was due to differences in the interactions between the individual proteins and ion-pairs with the O-H functional group found along the wall surfaces of the membrane channels The known zeta potential of alumina membrane is close to [29] and since the protein solution pHs were 5.5-7.5, the hydroxyl groups along the alumina wall surfaces should remain relatively unionized In contrast, at positive or negative transmembrane potentials, protein fluxes not differ significantly as at zero transmembrane potential in the single and mixed protein experiments for Lys and BSA This suggests that at high applied transmembrane potentials, the protein-protein interactions in the mixed protein experiments are less significant It is possible that the electric field within the nanochannels interacted strongly with the proteins and prevented the aggregation of proteins 3.3.3 An electrophoresis-potential shielding model for proteins’ transport in a single solution In the single protein experiments, the Lys and BSA fluxes increased at favourable transmembrane potentials compared to zero transmembrane 82 potential This could be explained by the electrophoretic transport mechanism for charged species according to the following equation: J =− zF δφ DC δx RT Eqn 3.1 where J is protein flux, D is diffusion coefficient, z is the charge of the protein molecule, x is the axial distance along the channel, φ is applied potential, C is protein concentration, F, R and T have their usual meanings Fig.3.4A shows the concentration profile of a charged protein in a membrane channel in which the protein molecules are attracted towards the receiver solution under the influence of a favourable transmembrane potential applied between both ends of the channel At the steady-state, it is expected that the protein flux arising from electrophoresis equals the diffusion flux across the membrane/receiver solution interface Interestingly, during the single protein experiments, the protein fluxes for all three proteins were observed to decrease significantly over time This suggested the driving force for the electrophoretic transport of the charged protein molecules decreased over time It is known that the potential of an electrode decreases exponentially across a double layer comprising 83 opposite-charged ions and this potential drop can be estimated according to Debye-Huckel approximation as follows: ϕ x = ϕ exp(−κx) Eqn 3.2 where κ is the inverse Debye length, φ0 is the applied electrode potential and φx is the electrode potential at axial distance x measured from the platinum layer electrode Fig 3.4 Schematics showing (A) the concentration profile of a charged protein transversing a membrane channel under the influence of a favourable transmembrane potential gradient; (B) the change in potential along the axial direction of the channel Diagrams depict the membrane/receiver end of a typical channel within the membrane E V is the working electrode potential applied at the electrode layer adjacent to the receiver solution lPt is the thickness of the platinum electrode layer which is ca 50 nm 84 During the transport experiment, the protein concentration in the receiver solution increased with time Due to shielding effect by the charged proteins, the electrical potential at the electrode/channel solution interface was expected to be considerably lower than the electrical potential applied at the electrode/receiver solution interface as shown schematically at Fig.3.4B Based on this model, the electrophoretic driving force decreases over time when the protein concentration in the receiver solution increases For example, the potential at the platinum/channel solution interface was estimated to decrease significantly to ca +0.02 V when the receiver concentration reached ca 4% of the feed concentration, which occurred experimentally ca after application of +1.5 V potential for the BSA single protein experiment The fluxes at initial time were similarly calculated with the additional consideration of the initial protein concentrations present at the electrode/channel solution interface after the pre-equilibration time Fig 3.5A shows the good agreement between the experimental data and theoretical flux values for BSA and Lys at favourable transmembrane potentials, calculated using Eqn 3.1 and Eqn 3.2 In addition, the mobilities of BSA and Lys derived from the initial experimental fluxes after consideration of the reduced 85 applied potentials at the electrode/channel interface [30], were 4.5×10-8 and 6.0×10-8 m2 s-1 V-1 respectively This was in good agreement with theoretical values calculated from the diffusion coefficients and protein charges using the Einstein relation Fig 3.5B shows the experimental data and theoretically calculated values for BSA and Lys fluxes over the duration of the single protein experiments for BSA and Lys at unfavorable transmembrane potentials It is obvious that the flux reversals at unfavorable transmembrane potentials predicted by electrophoretic driving force model were not observed experimentally The system is thus similar to the facilitated diffusion of solutes in cell membranes in which specific molecules transverse the membrane from high concentration to low concentration, through the action of specific carrier proteins [31] In our system, the selective transport was achieved based on differences in transport fluxes under the influence of a discriminating electrical potential gradient However, the electrophoresispotential shielding model could not adequately explain the protein fluxes during the mixed protein experiment, which were likely complicated by the possible interactions between the differently charged proteins 86 -2 -1 Flux ( μmol m s ) A 20 BSA (+1.5 V) 15 BSA (calculated) Lys (-1.5 V) 10 Lys (calculated) 0 10 20 30 40 50 60 Time (min) B BSA (-1.5 V) BSA (calculated) Lys (+1.5 V) Lys (calculated) -2 -1 Flux ( μmol m s ) 10 -5 -10 10 20 30 40 50 60 Time (min) -15 Fig 3.5 Plots of experimental and calculated fluxes of BSA and Lys proteins under the influence of (A) favourable and (B) unfavourable transmembrane potentials Charges of BSA and Lys used in calculations were -20 and +15 respectively, in solutions with pHs close to [32, 33] 3.4 CONLUSIONS We demonstrated the selective transport of three proteins by controlling the transmembrane electrical potential applied across a platinum-coated micrometer thickness nanoporous alumina membrane At the transmembrane potential of -1.5 V, the system was highly selective for Lys and achieved a 87 flux ratio of 96:1:12 for Lys:BSA:Mb in a protein mixture solution The system mimicked the facilitated transport of solutes in cell membranes in which a specific solute was selectively allowed to transverse the membrane down a concentration gradient, with enhanced flux compared to other solutes An electrophoresis-potential shielding model provided reasonable explanation of the decreasing experimental fluxes over time at favourable transmembrane potentials However, the model was inadequate in explaining the high fluxes observed at unfavorable transmembrane potentials and the lower fluxes observed in mixed protein experiments Further work is necessary to investigate the transport 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