Development of membrane based electrodes for electroanalytical applications 3

Several studies in the past thirty years have focused on constructing an artificial bilayer lipid membrane that can act as cell membranes [4, 5].. Unlike other works [19] in which the bi

3 CHAPTER 3

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

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 Ca ) were also embedded in 2+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”

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

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

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

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 2 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 kVm−1 suitable for electrokinetic transport of proteins using very small transmembrane potential of 2 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 7

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 are 1,4-N-acetylmuramidase, L-7001, N,O-diacetylmuramidase, PR1-Lysozyme, Globulin G1, Globulin G,

Lysozyme g, Mucopeptide N-acetylmuramoylhydrolase, 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

"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

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 5 g L−1, 2 g L−1 and 2 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 1

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 5 min 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 4

oC and used within three days of preparation 5 mL protein solution was

introduced into the feed compartment and the feed concentrations for BSA, Lys and Mb were 5 g L-1, 2 g L-1 and 2 g L-1, respectively As the receiver solution, 760 μL of ultrapure water was placed inside the cuvette cell Real-time absorbance of proteins was monitored at 280 nm, 410 nm or 600 nm

UV Detector Potentiostat

Permeate area

Membrane Holder Contact

Platinum

Platinum Platinum Layer

Alumina Membrane

Feed Solution

UV Detector Potentiostat

Permeate area

Membrane Holder Contact

Platinum

Platinum Platinum Layer

Alumina Membrane

Permeate area

Membrane Holder Contact

Platinum

Platinum Platinum Layer

Alumina Membrane

Feed Solution

Fig 3.1 Schematics of the biomimetic platinum-coated alumina membrane

system showing how the electrical potential gradient was applied across the micrometer-thick membrane housed within an electrically conductive holder

for Lys, Mb and dye-impregnated BSA, respectively, in single protein experiments All three proteins showed moderate absorbance intensity at 280

nm Sign convention of transmembrane potentials applied across the

membrane via surface contact was described for the receiver side, and measured with respect to the feed side

0 2 4 6 8 10 12 14 16 18

Fig 3.2 Effect of transmembrane potentials on the initial fluxes of BSA, Lys

and Mb in single protein experiments

Fig 3.2 showed the initial fluxes of proteins obtained from single protein experiments The protein fluxes across the membrane were obtained from the initial slopes of the protein concentration determined spectroscopically at the received side

of membrane with respect to experiment time It is clear from Fig 3.2 that Lys flux decreased as the transmembrane potential increased positively The high Lys fluxes were obtained at negative potentials, while the low fluxes occurred at zero potential and positive potentials The Lys protein was expected to be positively charged since its isoelectric point of Lys was 11.0, which was higher than the solution pH of 5.5 At

-1.5 V, the receiver side of membrane was of opposite charge to the Lys protein, thus the attractive electrophoretic force between the electrode layer adjacent to the receiver solution and the positively charged Lys protein increased its rate of movement across the membrane Interestingly, the reversed trend was not observed at positive potentials The Lys flux remained somewhat constant at positive potentials beyond zero potential This trend was different from the transport of positively charged bovine haemoglobin (Bhb) through gold–coated polycarbonate track etched

(PCTE) membranes reported by Chun et al [17] The transport of Bhb was highest

at zero potential but deceased with positive or negative applied potentials This phenomenon was explained by the surface roughness of the nanopore wall which formed high charged density regions and limited the transport of proteins due to strong electrostatic interactions This was attributed to the small pore dimensions (8.7 and 11 nm) which were comparable to the protein size (6 nm) In contrast, in our platinum-coated membrane, interactions of proteins with the charged platinum layer was confined to a thin platinum coating of ca 50 nm, overlying the porous alumina membrane Thus the direct interactions of proteins with the charged surface occurred over a much shorter

distance, unlike the 6 μm thick gold-coated polycarbonate membrane which

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 -1s , 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