Biotechnical and other applications of nanoporous membranes

Biotechnical and other applications of nanoporous membranes

Review Biotechnical and other applications of nanoporous membranes Pieter Stroeve and Nazar Ileri Department of Chemical Engineering and Materials Sc...

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Biotechnical and other applications of nanoporous membranes Pieter Stroeve and Nazar Ileri Department of Chemical Engineering and Materials Science, University of California Davis, Davis, CA 95618, USA

Recent advances mean that arrays of nearly uniform cylindrical, conical and pyramidal shaped pores can be produced in several types of substrates. Surface modification of nanopore surfaces can give unique mass transport characteristics that have recently been explored for biomolecule separation, detection and purification. Recent interest has focused on the use of nanoporous membranes for mass transfer diodes that act analogous to solid-state devices based on electron conduction. Asymmetric pores such as conical pores can show superior performance characteristics compared to traditional cylindrical pores in ion rectification. However, many phenomena for membranes with asymmetric pores still remain to be exploited in biomolecular separation, biosensing, microfluidics, logic gates, and energy harvesting and storage. Background Molecular transport controlled at the nanometer-scale using membranes offers great potential for high selectivity and high fluxes. Many applications, including protein separation and purification, biomolecule detection and drug delivery, are now being realized with nanoscale pore structures that can provide high selectivity based on specific molecular characteristics [1–3]. A key factor for high selectivity is enhanced molecule–pore interactions in nanopores. It is important to understand the effects of pore size and shape, pore surface modification, and possible osmotic flow and electric field variability within nanopores. Taken together, these parameters control the flux of biomacromolecules through nanopores. In the last 35 years or so, nanoporous membranes with a reasonably uniform pore-size distribution have become commercially available. Membranes with nanometer-scale features have many applications, such as in optics [4], electronics [5], catalysis [6], selective molecule separation [7–9], filtration and purification [10], biosensing [11–13], and single-molecule detection [14–16]. Physical filtration and molecular separation by membranes is not new. Indeed, nanopore–molecule interactions were described by experiments and theory after the development of tracketched membranes in the 1970s [17]. However, the evolution of nanotechnology has provided new opportunities for using smaller and more regular structures for porous membranes. Artificial sieves with higher precision and greater flexibility than track-etched membranes have been produced, with commensurate improvements in Corresponding author: Stroeve, P. ([email protected])

performance and functionality [17]. These new filters have facilitated the most detailed scientific investigations to date of membrane-performance-related phenomena. In addition, mesoporous materials, such as mesoporous nanoparticles, are useful for species detection, and for uptake and controlled release of biomolecules [18]. In this review we examine important developments in nanoporous membranes to improve transport and selectivity, with a focus on protein transport. Specifically, we discuss nanopore surface modification, control of transport in nanoporous membranes via external methods, protein separation, protein fouling, ion rectification and recent theoretical modeling of transport in nanoporous membranes. Nanoporous membranes Artificial nanoporous membranes are of current interest largely because of applications involving molecular sorting, sensing and separation [19,20]. For any emerging membrane technology, transition to commercial success requires both precise control over device performance and scalability of the membrane synthesis process [17]. Successful membrane synthesis processes should provide good control over the average pore diameter and produce a narrow distribution for pore diameter [21]. The membrane production process must also be economical. For many applications requiring extended functionality in harsh environments, membranes with mechanical, chemical and thermal stability are preferred. Biocompatible membranes with minimum fouling are also needed, especially for biomedical applications [19,21]. Table 1 summarizes membrane types, various fabrication methods and their advantages and disadvantages. The most common nanostructured filters are made of organic polymers. For example, polycarbonate track-etched (PCTE) membranes, produced by track etching in polycarbonate films, are available with pore diameters from 10 nm to 10 mm. Although the scalability of these membranes is good, uniformity and flow rates for 10-nm-diameter pores are limited to 20% and <0.1 mL min 1 cm 2, respectively (Sterlitech, The pore diameter variations and limited transport rates are often too low for many applications. Other porous filters, such as aluminum anodic oxide (AAO) and mesoporous silica, have been created via anodic etching of Al [22,23] and sol–gel processes [24], respectively. Pore dimensions of 30–400 nm can be obtained for AAO and 2–20 nm for sol gel films, but there is still a lack of uniformity and scalability. Conversely, membranes made of zeolites have very uniform pores, but

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Trends in Biotechnology June 2011, Vol. 29, No. 6

Table 1. Fabrication methods, their advantages and disadvantages for different type membranes Type of membrane

Chemical, Mechanical Stability, Biocompatibility Fair, Fair, Very Good

Organic polymeric membrane (polymeric membrane, e.g., PCTE) Good, Very Good, Good Inorganic membrane (e.g. AAO, and SiO2, Si, SiN)

Fabrication Method

Pore Morphol ogy

Uniformity Scalability Cost

Track etching Lithography

Ordered Ordered

Fair Good

Good Good

Relatively low costs for track etching

Anodization Sol-gel Microfabrication techniques

Ordered Tortuous Ordered

Good Fair Good

Fair Fair Good

Higher costs than organic membranes

only in a relatively narrow range of 0.3–3 nm (Lenntech, In addition to scalability and uniformity problems, commercial membranes are at least three orders of magnitude thicker than the diameter of biomolecules; increased membrane thickness leads to low transport rates and reduced size cutoff properties [8,9]. Hence, efficient fabrication strategies are still needed to create highly uniform, thin structures over mm2 areas. AAO and PCTE membranes AAO membranes are produced by anodic oxidation of an Al substrate in an aqueous solution of acidic electrolytes, such as oxalic, sulfuric, chromic and phosphoric acids [25]. The resulting membrane consists of closely packed hexagonal pores of 10–200 nm in diameter. The pore diameter depends on the type of electrolyte used and the anode voltage [26]; typical pore densities are of the order of 1011 pores cm 2 (Table 2) [25]. Pore length is typically tens of mm and cannot be readily shortened owing to the chemical mechanisms that give rise to nanopores. However, to achieve high throughputs and reduce permeate loss, membranes should be as thin as possible with adequate mechanical strength, because, according to Fick’s law or the Nernst–Planck equations, the mass transfer rate is inversely proportional to pore length [8,9,27]. Therefore, the thick nature of AAO membranes (60 mm) represents an important limitation in separation and filtration processes. Recently, Lee et al. reported fabrication of perfectly ordered alumina membranes with uniform pores of periodically modulated diameters via a hard anodization process that is much faster than conventional methods [28]. The process yields approximately three times higher interpore distances and approximately ten times higher pore densities. Although its thickness remains a limitation, the membrane provides high throughput. Diffusion and separation in AAO membranes have been studied by several groups. Kipke and Schmid, for example, studied the diffusion of crystal violet molecules encapsulated in sodium dodecylsulfate micelles, which revealed the suitability of AAO membranes for size-based sorting [29]. Modified AAO membranes have also been used for DNA immobilization, detection and separation [10]. Table 2. Typical properties of PCTE and AAO membranes Parameter Pore type Distribution Diameter Pore density (pores cm 2) Pore length (mm) 260

PCTE Monodisperse Random 10 nm–10 mm 10 9 3–11

AAO Narrow disperse Hexagonal 10–200 nm 10 11 60

More recently, a new fabrication technique for thin nanoporous aluminum membranes has been described and the electrostatic sieving effect of the membrane on protein separation was subsequently investigated [8]. PCTE membranes are manufactured by irradiating a polymeric sheet of desired thickness with accelerated heavy ions or high-energy alpha particles [30]. The transiting ions leave well-defined tracks of displaced atoms in the sheet that can be etched preferentially with an alkaline solution [31,32]. Completed PCTE membranes consist of randomly distributed cylindrical or conical pores with mostly uniform diameter ranging from 10 nm to 10 mm (Table 2). The highest achievable pore density is approximately 6  108 pores cm 2 (PCTE; [33–35]. AAO and PCTE membranes are relatively inexpensive, easy to fabricate and widely available. However, regardless of these attractive features, deficiencies in other properties, such as pore diameter uniformity, electrical conductivity, mechanical strength and biochemical stability, limit their integration into advanced nanodevices for many applications [17,21,36]. AAO membranes are thick and brittle, whereas PCTE membranes are thinner and robust. Surface treatment is usually necessary to change the mass transport rate and to impart improved biocompatibility. Membranes fabricated via lithographic techniques With recent advances in nanotechnology, several groups have demonstrated the fabrication of ordered cylindrical arrays of nanopores in inorganic materials such as silicon (Si) and silicon nitride (Si3N4) using lithographic techniques [7,9,37]. These processes have relatively high throughput, good control of pore diameter and are easy to integrate into microfluidic devices, which is important for future lab-on-a-chip applications. However, the production of these membranes is still costly [38,39]. Therefore there is a continuing need for inexpensive fabrication techniques that yield better-operating sieves. Devices fabricated with nanometer-scale features can facilitate direct dynamic manipulation of material properties at the molecular scale. Investigation of nanostructures and ensembles of nanostructures is therefore of great importance for many applications. For the majority of fluidic applications, membranes are required to have high transport rates, temperature and chemical stability, and good mechanical strength [40]. Furthermore, to facilitate biotechnological advances in protein screening and toxin or virus sensing, structures are needed with a uniform pore size and membranes with a large dynamic range of uniform pore diameter (1 nm to 1 mm).


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Filter properties such as thickness, permeability, stiffness and selectivity can be precisely controlled by means of microfabrication technology [40]. The standard lithographic techniques including focused ion beam (FIB) and electron beam lithography (EBL), which can create very small, arbitrarily shaped feature sizes with good uniformity and reproducibility, but at high costs and in small volumes. Photolithography is widely used, but its resolution is limited owing to diffraction [41]. Conversely, laser interference lithography is a simple method for creating periodic and quasi-periodic patterns [42] and is useful as a low-cost technique for the exposure of large areas. Feature sizes as low as half the wavelength of the light source used can be obtained. Such small, regular patterns are perfectly suited for microsieve production. For example, interferometric lithography has been used to manufacture Si3N4 filters with a pitch of 200 nm and a pore size of 65 nm [39]. Metallic sieves have also been fabricated using a combination of laser interference lithography and nickel electroplating, with 250-nm holes over a 1-cm2 area [43]. Most membranes today have cylindrical pores. However, sieves with conical or asymmetric pores offer advan[()TD$FIG]tages over the current technologies, such as improved (a)

molecular transport rates and greater control of selectivity. Conical nanopores fabricated in radiation-tracked polymeric membranes by chemical etching can rectify an ionic current similar to an electric diode [44]. Conical pores etched into the surface of a track-etched polymeric membrane using O2 plasma have demonstrated enhanced transport properties [35]. Finally, conical and double conical pores fabricated using ion track etching in ultrathin Si3N4 membranes have demonstrated higher fluxes [45]. For inorganic substrates, nanoporous membranes can be created by interferometric lithography, which can be adapted for large-scale production, and by EBL. The advantages of Si- and Si3N4-based filters over conventional sieves are increased chemical stability and biocompatibility and improved control over pore dimensions and surface properties. Arrays of conical- and pyramidal-shaped pores in Si wafers have been fabricated (Figure 1a) [46]. The authors demonstrated that interferometric lithography is a simple method for creating highly uniform, periodic patterns in a resist layer (Figure 1b), thereby resulting in improved pore uniformity compared to commercial membranes. They also characterized the transport behavior of the nanoporous membranes and found a 20-fold (b)

0.3 µm 0.5 µm

1.50 µm 5.0 µm


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Figure 1. (a) Scanning electron microscope images of a silicon membrane (wafer) with pyramidal-shaped nanopores. The view is from the larger opening on the front side to the smaller opening at the back side of the membrane. The inset shows a cross-section. (b) Scanning electron microscope images of a resist pattern defined by interferometric lithography. The inset shows a cross-section. (c) Simulation configurations for cylindrical, conical and pyramidal pores. Reproduced from [46] with permission.



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Table 3. Experimentally measured pyridine fluxes through nanoporous Si membranes versus commercial available PCTE membranesa Parameter


Pore size (nm) Membrane thickness (nm) Pore density (pores cm 2) Pyridine flux (mol cm 2 s 1)

100 6000 6  10 8 5.5  10

Si membrane conical pore, small apex pore = 100 nm, large pore = 250 nm 100 280 3  10 8 9.9  10 9



Modified from [46].

improvement in the flux of pyridine in the silicon membranes compared to PCTE membranes with the same pore size (Table 3). Whereas the pore size is the same in both the PCTE and Si membranes, the main advantage is the decrease in silicon membrane thickness. In Si membranes, arrays of pores can be fabricated in larger arrays of square patterns that are etched from the back side of the Si wafer to obtain very thin membranes [46]. Nanopore surface modification The surfaces of pores in nanoporous membranes can be modified for improved biomolecule separation. Self-assembled monolayers (SAMs) of alkanethiols provide a convenient way to attach functional groups to metal substrates such as gold, silver and copper that can be used in membrane technology, especially in nanofiltration [47]. Electroless deposition of Au onto the pore walls in PCTE filtration membranes has been reported for electrically tunable charge selectivity [48–50]. PCTE membranes contain well-defined cylindrical pores with a narrow pore size distribution; therefore, the surfaces of deposited Au provide an opportunity for further modification of membrane properties by chemisorption of SAMs on the inside surface of the pore walls. The pore size of Au-coated PCTE membranes can be controlled via the amount of Au deposited. Propanethiol has been chemisorbed to Au to protect the surface from adsorption of anions (e.g. Cl ) that could lead to excess negative charges on the pore walls, as well as irreversible changes in charge selectivity [49]. The authors reported introduction of permselectivity using such Au-coated PCTE membranes based on the hydrophobicity or hydrophilicity of functional tail groups (e.g. methyl or hydroxyl) of thiols chemisorbed to the gold [51]. Surface properties of Au-coated PCTE membranes have been modified using SAMs formed from weak

acid-functionalized thiols, and then used to prepare ionselective membranes with controllable pH-dependent permselectivity [52]. Such membranes are also very useful for controlled release of drugs and selective separation of proteins. The effect of surfactants on diffusion of atrazine and paraquat has recently been demonstrated using functionalized PCTE membranes, which revealed that the presence of adsorbed cationic and anionic surfactants could enhance or reduce molecular transport [53]. Nanopores with chemisorbed aptamers can achieve protein detection at single-molecule resolution [54]. The possibilities for modifying nanopore surfaces are extensive (Table 4). As demonstrated by the surface modification of mesoporous silica nanoparticles, unique mass transport and detection functions are possible because of the wide variety of chemicals available to modify the pore surface by physisorption or chemisorption [18]. External control of transport in nanoporous membranes Gold nanotubule membranes have attracted attention because they can be used to control ion transport selectivity by external means, for example by changing the pH and ionic strength and applying electrical potentials [55–57]. Electrostatic interactions between charged species and the charged pore wall can lead to ionic selectivity, depending on the thickness of the electrical double layer, the charge of the transported ions and the membrane surface charge. Furthermore, Au surface layers facilitate electrical contact with the Au nanotubules inside the membrane for control of the electrostatic potential [56]. Because the local pH and electrolyte concentration of the membrane solution dictate the ionization state of the membrane fixed charges [52] and the permeate ion [55], ion transport rates can be tuned using external solutions. It has been shown that the permeability to an ionic species of a charged nanoporous

Table 4. Surface modification methods and their applications Modification method Coating Molecular self-assembly

Chemical treatment Plasma treatment Surface graft polymerization Electroless deposition of metals


Comments Physical deposition of hydrophilic or biocompatible materials, not very stable Adsorption of SAMs or LBL assembly through chemical synthesis at the interface, chemically and thermally stable, simple Mostly oxidative surface treatment, introduction of functional groups by chemical means Use of plasma with oxygen UV and ionization radiation of the membrane surface for monomer grafting Deposition of gold, silver, copper, nickel and other metals in ultrathin layers, stable, metal surface can be further modified by chemisorption

Applications Medical devices, biomaterials Biosensors, SPR studies, bioseparation

Biosensors, drug delivery, bioseparation Bioseparation, sterilization, ocular prostheses, tissue culture BioMEMs, biomaterials Bioseparation, wastewater treatment, biomaterials

()TD$FIG][ Review

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6.0E-11 I = 0.01 M R = 11 nm


Key: BSA BHb


Flux of BHb pI = 7.0 I = 0.01 M R = 11 nm

5.0E-12 Flux (mole/cm2 S)

Flux (mole/cm2 s)






Key: BHb

3.0E-12 2.0E-12 1.0E-12 “off”







6 pH




1.0E-11 “off” “off” 0.0E+00 3



6 pH




TRENDS in Biotechnology

Figure 2. Comparison of flux vs pH for BSA and BHb transport with buffer solutions of I = 0.01 M. The insert shows a magnification of the BHb results. A change in pH has an off–on effect on transport, so that the nanopores act like mass transfer gates. Redrawn from [62].


der et al. fabricated ultrathin charged nanopores in graphene monolayers and investigated double-stranded DNA translocation [67]. Other authors have analyzed the diffusion of proteins in charged nanochannels for pH values above and below the isoelectric point for three proteins, and reported that electrostatic interactions dominate the process [68]. The diffusion coefficients reached maximum values when the proteins were neutral at their pI and pHcontrolled transport led to separation of biomolecules across the nanochannel [68]. The transition between surface-dominated and bulk diffusion regimes in nanochannels has recently been observed by controlling the pore charge [69]. pH effects on ion transport are also important in thick biological membranes, such as human skin [70]. Pores in 6.00E-13 BHb 5.00E-13

Flux (mole/cm2 s)

membrane changes by several orders of magnitude when the external pH is varied [55]. The transport of neutral and charged species under an electric field has been demonstrated [58]. Gold-plated, surface-modified nanotubes were used to separate proteins based on their diameter [59]. pH-switchable ion transport was demonstrated using weak-acid SAMs on Au-coated PCTE tubule walls [52]. A similar pH-switchable ion technique used chemisorbing cysteine on Au-coated nanopores inside PCTE membranes [60]. Ionic strength, pH and applied voltage effects on the selectivity of separation for similarly sized bovine hemoglobin (BHb) and bovine serum albumin (BSA) have been investigated [56,61,62]. For concentration-gradient-driven protein flux experiments at a low ionic strength of 0.01 M, BSA transport is 70-fold faster than that of BHb (Figure 2) [62]. By contrast, at an ionic strength of 0.1 M, the difference between BSA and BHb transport was reduced to a factor of 4.3 [61]. Other experiments have demonstrated that an applied electrical potential shifts the surface charge density of pores and can increase or decrease the molecular flux [56], as shown in Figure 3. In this figure, BSA has zero charge because it is at its pI and therefore is not affected by a change in pore surface charge due to different applied potentials. In comparison, BHb is charged and is sensitive to changes in surface charge on the pore walls, which leads to changes in the BHb flux. In another study, silica nanopore electrodes were used to impart transport selectivity via electrostatic control [63,64]. The effect of applied voltage on the selective transport of BSA, lysozyme and myoglobin was studied using platinum-coated nanoporous alumina membranes [65]. More recently, tapered nanopore-terminated probes of silica have been used for singlemolecule discrimination of chiral enantiomers [66]. Schnei-

pH = 4.7 I = 0.01 M

4.00E-13 3.00E-13 2.00E-13 1.00E-13 0.00E+00 -0.15 -0.1 -0.05







Applied potential (V vs. Ag/AgCl/3M NaCl) TRENDS in Biotechnology

Figure 3. Flux of BHb with applied potential at pH 4.7 and I = 0.01 M. Redrawn from [56].


Review the skin are charged, depending on the pH, and the pHdependent surface charge influences the ion flux. Pore fouling by proteins For micro-, ultra- and nanofiltration, protein fouling can lead to a loss in mass transfer performance of the membrane over time [71,72]. For protein separation, deposition of proteins within the nanopores can result in membrane fouling. To avoid protein fouling, the surface of Au-coated nanoporous membranes can be modified by chemisorption of a thiol-terminated poly(ethylene glycol) (PEG). Thiolterminated PEG can form a SAM on the surface of Au and has been used for planar and curved surfaces to prevent protein adsorption [73]. The thiol-terminated PEG is chemically bound to the Au surface and forms a hydrophilic layer that resists adsorption of proteins. Comparison of Au nanotube membranes with and without PEG modification revealed that membranes with PEG modification could be used for unblocked lysozyme protein transport over 5 days; conversely, the same membrane without PEG modification show blockage in only 8 h [59]. Surface-bound PEG can be present in addition to other SAMs that impart charge or change the surface tension of the nanopores. However, no fouling of the nanopores has been observed in repeated mass transport measurements of BHb and BSA [61]. Protein fouling could be more of a problem near the apex for conical nanopores (i.e. the smallest diameter). Conical nanopores are of interest for protein transport because the resistance to transport is less than for cylindrical pores. The resistance to mass transport is present mainly in the apex region, which is short. However, no protein transport studies have yet been performed with conically shaped pores. Microfluidics, diodes, logic gates and energy harvesting Controlled and precise fabrication of nanopores, selective modification of nanopore surfaces and the use of external control can lead to many new and unique applications other than protein separation and biomolecule detection. For example, tunable elastomeric nanochannels that facilitate reversible channel deformation have been fabricated for DNA manipulation [74]. Nanoporous membranes have been used for molecular gating of electroosmotic flow, with applications in nanofluidic and microfluidic devices [75]. Asymmetric nanopores, bipolar diodes, transistors and nanofluidics diodes with charged surfaces that are either uniform or have different regions of surface charge have been reported [76]. Many studies have focused on the ionic flux, electrical current and rectification properties of asymmetric nanopores [77,78]. Analytical solutions on ion transport in asymmetric pores show a range of rectification properties depending on pore shape and size, surface modification and the effect of external parameters, such as pH and ionic strength, which control the charge and charge distribution of the surface of the pores [79,80]. Optimization of asymmetric nanopore characteristics is important for nanofluidics applications. Ali et al. have extensively modeled nanostructure characteristics and investigated new experimental methods to tune nanopore selectivity and electrical current for practical applications such as logic gates using nanofluidics diodes based on 264

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conical nanopores with functionalized surfaces [81]. In principle, the electrical current induced by an ionic gradient across conical nanopores can be used to harvest the energy associated with the concentration difference [82]. This suggests that by imparting electrical energy to cause a concentration difference in ions across the nanoporous membrane and then recovering (i.e. re-harvesting) the energy, the nanoporous membrane system can be operated as an ionic battery to store energy; this might be useful for powering measurement devices in biological applications. Controlled release of drugs Nanoporous membranes provide a unique system for controlling the transport rate of not only proteins and other biological molecules, but also drugs. Surface modification, including the surface at the pore mouth, can have profound influences on the transport rate of specific drugs. Nanoporous membranes, nanoparticles, microparticles and microcapsules have been explored as media for the controlled release of drugs. Micro- and nanoparticles of mesoporous silica (and to a lesser extent mesoporous silica), prepared using sol–gel techniques, are of special interest in that the pore size is small, the pore structure is uniform and surface modifications at the pore mouth on particle surfaces have been extensively studied [18,83–85]. The surface chemistry of silica modification has been well developed for controlling mass transport of drugs, and biocompatibility can also be imparted to the silica surface [18]. Unique phenomena such as photochemically controlled gating of mass transport have been devised [85]. Molecular dynamics simulation of transport in nanopores Asymmetric membranes such as conical membranes have attracted considerable interest because of unique transport properties [86]. Molecular dynamics (MD) simulation can be used to study molecule transport (e.g. water, ions, dissolved gas, glycerol and many other small molecules) through biological membranes [87–93]. MD simulations are important for exploring protein transport on the nanoscale, in which the size of protein molecules is close to the size of the nanopores. However, diffusion of proteins through nanoporous membranes with cylindrical and conical pores has only recently been investigated [46]. For MD simulations, a solvent-free coarse-grained model was used in which proteins were treated as colloidal particles. Three different pore geometries, cylindrical, conical and pyramidal (Figure 1c), were studied in large-scale ESPResSo (extensible simulation package for research on soft matter) simulations. Higher diffusion rates were obtained with tapered geometries compared to the cylindrical geometry. The results can be explained by the much greater wall hindrance for cylindrical pores in comparison with tapered pores. Ionic current rectification and ion dynamics at different pH conditions in silica and PET nanopores have been investigated using atomic-level MD simulations [94,95]. In both studies, the NAMD program was used to simulate systems consisting of silica or PET, water and ions. Ionbinding spots on silica nanopores changed the ion concentration and resulted in ionic current rectification [94]. The

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