Biotechnology Advances 30 (2012) 1089–1099
Contents lists available at SciVerse ScienceDirect
Biotechnology Advances
j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / b i o t e c h a d v
Research review paper
Where bio meets nano: The many uses for nanoporous aluminum
oxide in biotechnology
Colin J. Ingham a, b,⁎, Jurjen ter Maat a, c, Willem M. de Vos b
a
b
c
MicroDish BV, Utrecht, Netherlands
Laboratory of Microbiology, Wageningen University, Wageningen, Netherlands
Laboratory of Organic Chemistry, Wageningen University, Wageningen, Netherlands
a r t i c l e
i n f o
Available online 12 August 2011
Keywords:
Anodized alumina
High throughput screening
Cell culture
Microbiology
Label-free imaging
a b s t r a c t
Porous aluminum oxide (PAO) is a ceramic formed by an anodization process of pure aluminum that enables
the controllable assembly of exceptionally dense and regular nanopores in a planar membrane. As a
consequence, PAO has a high porosity, nanopores with high aspect ratio, biocompatibility and the potential for
high sensitivity imaging and diverse surface modifications. These properties have made this unusual material
attractive to a disparate set of applications. This review examines how the structure and properties of PAO
connect with its present and potential uses within research and biotechnology. The role of PAO is covered in
areas including microbiology, mammalian cell culture, sensitive detection methods, microarrays and other
molecular assays, and in creating new nanostructures with further uses within biology.
© 2011 Elsevier Inc. All rights reserved.
Contents
1.
2.
3.
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . .
Origins and fabrication of PAO . . . . . . . . . . . . . . . . . .
PAO in microbiology . . . . . . . . . . . . . . . . . . . . . . .
3.1.
Counting and cell identification . . . . . . . . . . . . . .
3.2.
Growth and microcolony imaging of microorganisms on PAO
3.3.
High throughput microbiology . . . . . . . . . . . . . . .
4.
Eukaryotic cell culture . . . . . . . . . . . . . . . . . . . . . .
5.
Surface modification and functionalization of PAO . . . . . . . . .
6.
Physical detection technologies . . . . . . . . . . . . . . . . . .
7.
Indirect uses — PAO as the basis for fabricating other nanostructures
8.
Future prospects . . . . . . . . . . . . . . . . . . . . . . . . .
Acknowledgments. . . . . . . . . . . . . . . . . . . . . . . . . . .
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
1. Introduction
Nanoporous membranes can have unusual or extreme properties,
which have attracted intense interest from nanotechnologists and
Abbreviations: MALDI-MS, matrix-assisted laser desorption/ionization mass spectroscopy; PAO, porous aluminum oxide; PDMS, polydimethylsiloxane; PEG, polyethylene glycol; POP, proof of principle; SERS, surface enhanced Raman spectroscopy; SPR,
surface plasmon resistance.
⁎ Corresponding author at: Laboratory of Microbiology, Dreijenplein 10, Wageningen
University, 6703 HB Wageningen, Netherlands.
E-mail addresses: [email protected], [email protected] (C.J. Ingham).
0734-9750/$ – see front matter © 2011 Elsevier Inc. All rights reserved.
doi:10.1016/j.biotechadv.2011.08.005
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1089
1090
1092
1092
1092
1093
1093
1094
1095
1096
1097
1097
1097
material scientists, and which have also resulted in applications in
quite diverse sectors of biotechnology. Such materials can have an
exceptionally high surface area — at least 2 to 3 orders of magnitude
greater than planar surfaces. A high surface area has major advantages
in chemical catalysis, bioreactors and displaying arrays of detector
molecules. Nanopores have other attractive properties: narrow pores
with high aspect ratios (pore diameter vs pore length) (Bolton et al.,
2011) can act as selective portals in a way that mimics the way pores
in cells sense or take up specific compounds and can be effective
supports for chemical purifications and separations. Also, highly
ordered nanoscale surfaces can greatly enhance some optical and
physical detection methods (e.g. optical wave guides, surfaced
enhanced Raman spectroscopy). Finally, those nanoporous materials
1090
C.J. Ingham et al. / Biotechnology Advances 30 (2012) 1089–1099
that do exist can be used as templates to create other nanoscale
materials with further useful properties.
The variety and ingenuity of fabricated nanoporous membranes is
growing. These materials include fibrous meshes such as nanocellulose and other organic polymer filters, some of which are produced by
processes taken from the textile industry such as electrospinning
(Poinern et al., 2011). Self-ordering block copolymers have also been
used to fabricate nanoporous membranes (Bolton et al., 2011). Other
attractive types include nanoporous materials of silica/glass and a
range of metal foams and metal oxides. The latter group includes
oxides capable of self-assembling into nanoporous structures under
anodizing conditions and can be formed from metals such as titanium,
tin and aluminum oxides. The nanoporous forms of aluminum oxide
have long attracted the attention of materials scientists and electrochemists. Porous aluminum oxide (PAO; or anodic aluminum oxide
(AAO) or porous anodic alumina (PAA)) is now finding many uses at
the intersection between nanotechnology and biotechnology. In part,
the widespread application of PAO in biotechnology is related to the
controllability of fabrication. Remarkably well-ordered, consistent
and regular arrays of pores with a high aspect ratio can be formed by
self-assembly when an appropriate voltage is applied to aluminum.
The resulting PAO has high aspect ratios, with deep and narrow pores,
that are often beautifully ordered, i.e. features that polymeric membranes or other manufacturing processes struggle to match. Moreover, aluminum oxide is inert and biocompatible. Aluminum is one of
the lowest valency metals not commonly found in life, the oxide is
highly insoluble and, unlike many other aluminum salts, non-toxic
which results in excellent biocompatibility. PAO has a low background
with respect to many detection techniques, including fluorescence
microscopy. Additionally, aluminum oxide does not change volume
greatly with temperature changes or wetting, is compatible with a
wide range of solvents and is exceptionally thermostable (some forms
withstand temperatures in excess of 1000 °C). The manufacture of
nanoporous metal oxides is not recent, and the availability of commercial forms over the last few decades may have contributed to the
wide range of ingenious applications. To date over 500 patents have
been filed on the manufacture or use of anodic alumina (source —
European Patent Office, searches for anodic alumina and anodic
aluminum oxide).
2. Origins and fabrication of PAO
An industrial process for the production of pure aluminum has only
existed for around 150 years (Edwards et al. 1930). Fifty years before
that time the metallic element was known (and indeed sometimes
prized above gold) but was not widely accessible. Aluminum is an
interesting metal for its lightness, abundance and anticorrosion properties. Resistance to corrosion is primarily due to passivation, the
formation of an inert and protective oxide layer on the surface. This
native oxide layer can be made more substantial by anodization, an
electrochemical oxidative process, which creates resistant non-porous
surfaces when performed using neutral electrolytes (Keller et al.,
1953). Both the lightness and corrosion resistance were drivers for the
introduction of this material to protect components in seaplane
manufacture (Lee, 2010). Over the past century PAO has been
developed by commercial organizations and academics (major
advances are summarized in Table 1).
When the neutral electrolyte is replaced with an acidic one, such as
phosphoric acid, the oxidative process produces an array of semiordered parallel nanopores with a closed base (Keller et al., 1953). The
structural features of this oxide (interpore distance, pore diameter
and barrier thickness) are strongly dependent on the anodization
voltage, the operation window of which is determined by the choice
of electrolyte. For example, anodization in phosphoric acid requires
higher voltages and yields larger pores than anodization in sulfuric
acid. The choice of electrolyte also affects the chemical composition of
Table 1
Key developments in PAO manufacturing.
Year
1827
Advance
Fredrick Wohler isolated pure alumium metal (Hans
Ørsted partially credited 1925)
1856
Henri Deville develops the first industrial method for
aluminum production
1923
Anodization of alumium for the protection of seaplane
components from corrosion.
1953
Effect of acidicelectrolyte and voltage on controlling pore
size described
1989
Publication of controlled manufacturing process
resulting in porous membrane
1999– Improved versions (pore regularity, new processes)
2011 driven by nanotechnology and semiconductor industries
Reference
Edwards et al.
(1930)
Deville (1859)
Lee (2010)
Keller et al.
(1953)
Furneaux et al.
(1989)
Lee (2010)
the PAO, as acid anions are incorporated into the oxide during
anodization (Thompson and Wood, 1981). This can be expected to
have significant effects on the mechanical properties of the PAO. It
appears unlikely that anions that contaminate PAO will be available as
nutrients for cell growth. However, there will also be effects on the
charge and chemical reactivity of PAO that may be relevant to cell
attachment and functional modification of the alumina. Other parameters that are important during anodization are temperature (Aerts
et al., 2010; O'Sullivan and Wood, 1970) and bath agitation.
A major advance, originating from the aluminum manufacturer
Alcan, was to progressively reduce the anodizing voltage during
manufacture (Furneaux et al., 1989). This results in detachment of the
oxide film from the underlying aluminum, yielding a porous membrane. During detachment, the barrier layer in contact with the aluminum plate thins, so that for a few microns of depth individual pores
are narrowed and subdivided (Figs. 1 and 2). This results in the most
widely available commercial form of PAO, which has clear asymmetry
between the two sides. Properties of commercially available formats
are displayed in Table 2. These formats are mainly sold for the
purposes of filtration (e.g. Whatman/General Electric Anotop and
Anodisk filters, Table 2) and mammalian tissue culture (e.g. Nunc Cell
Culture multiwell plates).
Under specific anodization conditions, prolonged oxidation results
in self-organization of the pores, thereby creating highly ordered
structures (Masuda and Fukuda, 1995). This has led to the development of a two-step oxidation process, where a sacrificial oxidation
layer is formed until the oxidation reaches a steady state. Removal of
the oxide layer leaves the underlying aluminum imprinted with the
periodic structure, from which a well-ordered porous structure is
directly formed during the subsequent oxidation step (Masuda and
Satoh, 1996). PAO formed under these self-ordering conditions has a
porosity of about 10% (Nielsch et al., 2002) and has defect-free areas up
to several microns in size. The requirement for a constant voltage does
not allow lift-off using progressive voltage reduction. Therefore,
well-ordered PAO is usually manufactured from thin aluminum
films. After anodization, the substrate is etched, e.g. with an aqueous
CuCl2–HCl solution. Alternatively, etching can be performed with
saturated HgCl2 followed by 5% phosphoric acid. These processes
remove the remainder of the aluminum and open the pores to produce
a symmetrical membrane.
The limited number of known self-ordering conditions puts restrictions on the available membrane formats. More flexibility is
introduced by premolding the aluminum film with an imprint stamp
(Masuda et al., 1997). During subsequent anodization, the imprinted
pattern is reproduced in the oxide, resulting in exceptionally ordered
arrays (Fig. 1). However, the maximum aspect ratio of well-ordered
pores that can be obtained after the imprinting of deviating patterns
remains limited (Asoh et al., 2001).
In recent years it has been shown that self-ordering regimes also can
be created at high anodization voltages (Lee et al., 2006). Anodization
outside the operation window of mild anodization can result in
C.J. Ingham et al. / Biotechnology Advances 30 (2012) 1089–1099
Fig. 1. A. High resolution SEM of surface of commercial PAO (Anopore), with pores
ranging from 200 to 40 nm in diameter. B. Lower resolution SEM of same surface, with
the nanopores narrowed by detachment from the aluminum block during manufacture.
C. Reverse of panels A and B with pore diameters from 100 to 200 nm. D. Surface as
panels A and B with upper surface sheared off in central region revealing structure
beneath, showing how pores are subdivided in the upper few microns. E. More highly
ordered and porous PAO with pores around 300 nm diameter created by nanoimprinting with hexagonal arrays of nanopillars (120 nm width, 250 nm pitch) followed by
anodization (Kustandi et al., 2010). F. Similar to panel E except an array of square
nanopillars (100 nm width, 200 nm pitch) was used for imprinting to create pores of c.
250 nm across. G. PAO with 40 nm pores lined with 3 nm silicon nanotubes (Lee et al.,
2002). Scale bar in panel G indicates the following: 80 nm when applied to panel
A; 350 nm panels B and C; 700 nm panel D; 550 nm panels E and F; 120 nm panel G.
breakdown of the oxide film (Ono et al., 2004), but when an oxide layer
of N400 nm was preformed, anodization at higher voltages proved to be
stable. This hard anodization process yielded well-ordered structures in
a relatively wide operation regime. The elevated voltage also results in
an increased anodization rate (N50 μm/h compared to 2–6 μm/h for
mild anodization). On the other hand, the porosity obtained was
reduced to only 3.3%.
The availability of different anodization regimes has led to the
fabrication of more advanced structures (Lee, 2010). By alternating mild
and hard anodization, an oxide with an alternating pore diameter could
be obtained (Lee, 2010). The formation of serrated pores was also
demonstrated in another advanced fabrication method (Li et al., 2010).
Gradients of different diameter nanopores over distances up to a centimeter were obtained using asymmetric voltages (Kant et al., 2010). By
combining anodic oxidation with selective aluminum etching, very thin
PAO membranes that were supported by unetched aluminum were
obtained, these membranes showed enhanced flow rates during filtration (Thormann et al., 2007). Finally, composites between PAO and
other materials and surface chemistry modifications that complement
the properties of the base material are also emerging (e.g. Choi et al.,
2010; Lee et al., 2002; Pera et al., 2010).
Whereas much empirical knowledge exists on the formation of
PAO, the mechanism of pore formation is still not fully understood.
Pore formation has been explained by the combination of electrochemical oxidation with field-assisted oxide dissolution (Hoar and
Mott, 1959; O'Sullivan and Wood, 1970), as well as the direct injection
of Al 3+ ions into the solution (Siejka and Ortega, 1977). To describe
the ordering phenomena, models that are based on mechanical stress
1091
Fig. 2. Manufacture of PAO. Illustration of asymmetric PAO manufacture. A. The starting
material, exceptionally pure aluminum (N99.99%) with a natural layer of oxide.
B. Further oxidation and formation of self-ordering pores during the anodization
process. C. Detachment from the aluminum block leading to an asymmetric pore
structure. The aluminum block is then free for creation of more PAO membranes.
formation (Jessensky et al., 1998; Li et al., 1998) and, more recently,
on equal field strength were introduced (Su and Zhou, 2008). Alternatively, pore formation has also been related to oxygen evolution
(and atmospheric pressure) during anodization, something not
adequately explained by other theories of PAO formation (Yang
et al. 2009; Zhu et al., 2008).
A drawback of PAO is that it is relatively brittle: it is a strong
material relative to its mass but thin sections with high porosity can
be vulnerable to breakage. Care must be taken to support or reinforce
large sheets. PAO can be cut using a laser or precise high-pressure
water jet and sterlized by plasma, dry heat, irradiation or solvents. A
side effect of PAO manufacture, sometimes seen on commercial PAO,
is a phenomenon called “hot spots” which are intense points of
fluorescence on an otherwise low background material. In other
contexts, such as gold or silver coated PAO, deliberately created hot
spots may be a useful image enhancement technique (Qiu et al.,
2010).
The intention of the remainder of this review is to examine the role
of PAO membranes within biotechnology (Fig. 3 gives key examples),
Table 2
Properties of commercially available PAO formats.
Property
Range
Pore size
Thickness
Porosity
Pore density
Size
20–200 nm
50–100 μm
25–50%
109–1010 cm− 2
13, 25 or 45 mm diameter circles
1092
C.J. Ingham et al. / Biotechnology Advances 30 (2012) 1089–1099
paying particular attention to the exploitation of this material for cell
culture and imaging over the last two decades.
phi29 packaging nanomotors on PAO as part of a hybrid bio-nano
structured device.
3.2. Growth and microcolony imaging of microorganisms on PAO
3. PAO in microbiology
3.1. Counting and cell identification
Counting microorganisms, stained for greater contrast, on the
surface of a microporous membrane by microscopy is a common
activity. Relatively rapidly after the widespread availability of commercial asymmetric PAO membranes, these were adapted for bacterial counting (Jones et al., 1989; Williamson and Palframan, 1989)
using acridine orange or ethidium bromide as fluorogenic dyes with
counting by fluorescence microscopy. Improved sensitivity over
flexible polymer membranes was reported, attributed to a low autofluorescence background of the alumina and the ease of focus on the
microorganisms filtered onto the rigid, flat upper surface of the PAO.
Subsequent work on plankton supported these conclusions and suggested applications in presenting microbial samples for scanning
electron microscopy (McKenzie et al., 1992). Additionally, fluorescence in situ hybridization has been performed successfully on PAO
(Ramsing et al., 1996). The low background properties were also used
to capture microorganisms for fluorogenic Gram-staining from cerebrospinal fluid (Durtshi et al., 2005a) with N10-fold sensitivity
compared to polycarbonate membranes. This technique increased
the contrast obtained by fluorogenic staining on PAO by blackening
the alumina with nickel nanoparticles (Durtshi et al., 2005b). Coating
the PAO surface with patterned silver nanocap arrays is also known to
enhance fluorescence microscopy of cells (Qiu et al., 2010). This is
believed to work by transferring plasmon resonance energy from the
silver nanocaps to the nearby fluorescent molecules.
Non-fluorescence sensing methods (covered more fully in Section 6)
have also been used for the quantification of microorganisms. Infrared
spectroscopy has been used to quantify bacterial spores, taking
advantage of the good IR imaging properties of gold-coated PAO and
also the even distribution of spores filtered onto the surface (Schiza
et al., 2005).
In the above assays the pore size of all forms of PAO is adequate to
act as a sterile filter and therefore capture all cells for counting. The
precision to which the pore size can be fine-tuned has also been used
for selective capture of bacteriophage phi29. By engineering a precise
pore diameter of 40 nm Moon et al. (2009) managed to capture intact
viral particles and array them within the pores of the PAO, while viral
capsids lacking DNA could be passaged through the alumina membrane. This technique has applications in analysis of viral assembly,
screening for variants in assembly and the possibility of arraying
The methods described in the previous section are all performed on
microorganisms captured without any attempt to culture them on the
PAO surface. Subsequently, microcolony growth and imaging on PAO
has been developed. This effort has been primarily directed at rapid
antibiotic sensitivity testing of bacterial pathogens and in studies of
microbial population heterogeneity, particularly in response to the
imposition and recovery from stress.
To date PAO has been shown to culture any organism tested that
can be grown in a Petri dish on agar. Tests on bacterial growth indicate
that growth on PAO supplied with the appropriate nutrients from
beneath is as rapid as in liquid medium (den Besten et al., 2007, 2010;
den Hertog et al., 2010; Ingham et al., 2005, 2008b). Additionally,
many microorganisms that cannot be grown on conventional growth
media can be cultured on PAO. One such group includes organisms for
which agar is either impractical or toxic. An example is the growth of
the Archaeal extremophile Sulfolobus sulfotaricus at 80 °C and pH b 4
on PAO, conditions under which agar cannot remain gelled, but which
are not a problem when using aluminum oxide as a matrix. Also,
porous culture supports can be used to grow microbes in close
communication with their natural habitat. This results in higher
culturability for a variety of reasons, including the presence of an
appropriate level and type of nutrients, and the presence of microbially secreted products such as siderophores and quorum sensing
molecules that influence the growth of other species (Ferrari et al.,
2005; Kaeberlein et al., 2002). This type of technique also provides the
ability to image microcolonies, enabling the detection of very slow
growing organisms or ones which will not form visible macroscopic
colonies. PAO has formed at least a component of porous culture
systems that have been used to screen for new species of soil and river
bacteria (Ferrari et al., 2005; Ingham et al., 2007).
One group of applications is microcolony based antibiotic susceptibility tests based upon culture of clinical isolates on PAO with
and without antibiotics, staining with fluorogenic dyes and image
analysis have been devised. Such tests have been applied to rapidly
growing pathogens such as the Enterobacteriaceae and methicillin
resistant Staphylococcus aureus, producing results within hours
(Ingham et al., 2006; Tsou et al., 2010). Greater value is added for
slow growing organisms, with susceptibility testing for Mycobacterium
tuberculosis reduced from over 10 days to 3 days (Ingham et al., 2008a).
An interesting alternative for M. tuberculosis is real-time monitoring of
growing microcolonies, allowing the effect of the addition or removal of
drugs during culture to be assessed (den Hertog et al., 2010).
Fig. 3. Key applications and developments of PAO in biotechnology. Important advances in green/red/blue and white biotech and in related areas.
C.J. Ingham et al. / Biotechnology Advances 30 (2012) 1089–1099
A second area of using microcolony culture and imaging (Durtschi
et al., 2005c) on PAO is in investigating phenotypic heterogeneity. The
ability to analyze large numbers of cells has facilitated quantification
of subpopulations. As with antibiotic susceptibility testing, culture on
PAO that can be moved between different Petri dishes allows stresses
to be imposed and removed. Culture under extreme conditions where
growth is marginal and minimal has been used to assess responses to
high salt (den Besten et al., 2007), low temperatures (den Besten et al.,
2010) and low pH (Ingham et al., 2008b).
PAO has also been used to investigate the communication and
competition between different strains of the swarming bacterium
Proteus mirabilis (Budding et al., 2009). In this case, the selectivity of
permeation of signaling molecules though the controlled pores was an
important issue. The controllability of permeation through PAO pores,
via modifying size and/or surface chemistry, are exploitable features
and will be further discussed in Sections 4 and 5.
3.3. High throughput microbiology
A logical extension of microbial culture on unstructured PAO is to
subdivide different areas of the surface to create discrete growth
compartments with a PAO base (Fig. 4). Given the small size of
microcolonies that can be effectively imaged the sample density can
be extremely high. In conjunction with other components, such as an
imaging and cell recovery platform, this allows high throughput.
Printed polymers allow medium to low sample density (Ingham et al.,
2005) while microengineering processes can be used to create culture
chips with an unprecedented number of discrete culture areas:
1 million in an 8 × 36 mm area (Ingham et al., 2007). This is part of a
trend within microbiology, of accelerating miniaturization of culture
formats that is a similar to Moore's Law that describes an exponential
growth in computing power (Gefen and Balaban, 2008). Uses of
culture chips to date have included viable counting, using the grid
pattern of the microwells to facilitate software recognition of microcolonies. High throughput screenings using fluorogenic substrates to
detect enzymatic activities of interest have been completed (Ingham
et al., 2007). The porous base has permitted culture on near-natural
Fig. 4. High throughput screening culture chip. A. Illustration of culture chip with a grid
pattern of micron-scale square compartments with a PAO base. Green and yellow
microcolonies of bacteria are shown. B. Array of microcolonies growing in 20 × 20 μm
culture wells detected by fluorescence microscopy. Visible are over a thousand
microcolonies (white, each containing N50 bacteria) from a chip with 180,000 culture
areas. C. Single well from alternative version of a culture chip showing a single180 μm
diameter culture area with bacteria labeled with the fluorogenic dye Syto 9 and imaged
by fluorescence microscopy. D. SEM of same diameter culture well as panel C, viewed
from above at a 45 degree angle. Illustration of printing method with PDMS stamp
dropped from a few microns hight to ensure even contact with the PAO beneath. Shown
are bacteria with quantum dots used to label the surface. F. PDMS pin loaded with
fungal spores (colored green). G. Image of 40 × 40 μm compartments (3 × 5 are shown)
printed with bacteria using a PDMS stamp, the bacteria were labeled with Syto 9 before
printing and visualized by fluorescence microscopy.
1093
substrates (sediment, river water, fecal material) as the growth
media. As with other culture systems that use natural materials separated from the samples (Ferrari et al., 2005; Kaeberlein et al., 2002)
this system permits the culture of microbes that have not been
successfully grown in defined media. Such a strategy, combined with
phenotyping, has value in isolation of new microbes and in the
creation of metagenomics libraries.
A high-density microbial culture format requires complementary
technologies for full functionality. A high throughput contact printing
method has been developed using an array of thousands of polydimethylsiloxane (PDMS) pins to print microorganisms within
40 × 40 μm wells (Fig. 4). The PDMS stamp is targeted and deployed
using a microscope, imaging through the transparent PDMS (Ingham
et al. 2010). This technique can print thousands of microcolonies on
planar PAO and hundreds on culture chips, the main limitation for the
latter being the precision to which the PDMS stamp can be aligned with
the target wells. An advantage of a physical stamp is that it can be used to
print replicas in multiple culture chips; this may be useful in the storage
of strain libraries or in screening processes. It is likely that non-contact
printing of bacteria (Zheng et al. 2011) is also possible within wells of a
culture chip. Additionally, low cost microscopy and imaging supports
this type of approach (Albeanu et al., 2008; den Hertog et al., 2010;
London et al., 2010).
4. Eukaryotic cell culture
The culture of mammalian cells is also possible on PAO. This is
commonly performed in co-culture systems where two cell types are
separated by a PAO membrane. Commercial tissue culture inserts
generally fit from 6 to 96 well microtiter plates. One advantage is, as for
bacteria, clarity of imaging by fluorescence microscopy compared to
more autofluorescent microporous membranes. Additionally, the
precision to which the nanopores can be engineered allows large
molecular complexes or subcellular aggregates to be excluded with
greater confidence than for less consistent membranes. A relevant
example of cell culture is that used to study communication between
malarially infected and uninfected human blood cell populations
separated by a 20 nm diameter pore PAO membrane (Schlozen et al.,
2009). Apart from separation of the two cell populations (for which
purpose other porous membranes would suffice) the precise and fine
pore size had two functions: (a) to exclude the transfer of hemozoin
crystals (a method of disposal of otherwise toxic hem by the malaria
parasite) from the infected to uninfected cell populations and (b) to
exclude transfer of major histocompatibility complex class II exosomes
which could have otherwise mediated antigen transfer between the
two cell populations.
Part of the attraction of PAO is in creating scaffolds for the development of cultured cells in a desired direction. This may be simply
through alterations in texture (pore size, spacing) or more sophisticated
surface functionalizations that recruit specific cell types to precise
locations or induce particular types of development and growth.
Engineered cell platforms intended for screening purposes and
based around PAO have been developed. Broadly, the approach of
subdividing the material into microwells with PAO at the base has
been implemented for mammalian culture, as for microbiology. For
example, microwells have been created using a polyethylene glycol
(PEG) hydrogel (Yu et al., 2009). This was achieved by silanating the
PAO surface followed by photolithography using a shadow mask to
polymerize the PEG in the desired pattern. The detection method
(impedence) used to read out changes in cell growth with anticancer
drugs is described in Section 5.
Beyond simple cell growth, i.e. single cell types or cocultures separated by a PAO membrane, lies tissue engineering: the engineered
manufacture of tissues and organs for research and transplantation.
Cells grown on an appropriate 3D scaffold and supplied with nutrients
and growth factors behave very differently to those grown by
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conventional cell culture methods on planar plastics or glass. Nonplanar scaffolds are often far better at encouraging cell growth that
more realistically mimics what is happening within the human body.
Alumina was first suggested as a suitable transplantation material for
hip replacements (Hamadouche et al., 2002). Nanoporous alumina
allows fine-tuning of cellular responses by altering surface properties
and the selective delivery of small compounds and larger aggregates
of biomolecules (Schlozen et al., 2009). Organic polymers have a
significant role in tissue engineering too; biodegradability and the
ability to mimic the mechanical properties of the tissue are attractive
features. However, nanoporous ceramics and other inorganic supports
can be less inflammatory (Poinern et al., 2011). Bone cell (osteoblast)
culture has been achieved on PAO — indeed PAO appears as an
excellent scaffold that is close to the porous nature of the surfaces that
exist in the body. While aluminum ions have been reported to have
negative effects on some types of bone development, this does not
seem to be a significant difficulty in this situation (Poinern et al.,
2011). PAO that is more flexible than that used in bone culture, to
allow molding of the shape of the tissue to body surfaces, is also being
investigated for culture of skin tissue for subsequent transplantation
(Parkinson et al., 2009). For artificial tissue engineering the PAO may
not necessarily be introduced into the human body, although this is
possible in some scenarios. PAO is also used in creating implantable
drug delivery systems with low inflammation potential (Gultepe
et al., 2010). If phosphoric acid is used in the anodization process then
the remaining phosphate incorporated into the alumina surface may
improve biomineralization. Immunosuppressive drugs have been
delivered in vivo at appropriate doses using cardiovascular implants
of drug-loaded PAO in rabbits, in addition to supportive in vitro
studies on drug release from this material (Wieneke et al., 2003).
Some tissue transplantation may require selective permeability —
for example to permit low molecular weight compounds to reach the
cell but to protect the newly grafted tissue from the host immune
system. PAOs with controlled permeability due to functionalization
with polyethylene oxide have been created for the immunoisolation
of tissue transplants (Lee et al., 2011). Such modified membranes
exclude antibodies but permit access of insulin and glucose and
therefore may be suitable for the encapsulation of pancreatic islet cells
for treatment of type I diabetes.
In addition to the use of PAO as a direct scaffold for cell, it is
possible to create structures using PAO as a template; such nanowire
arrays have significant uses in tissue engineering. These are discussed
in Section 7 (Indirect uses — PAO as the basis for fabricating other
nanostructures).
5. Surface modification and functionalization of PAO
Coupling other molecules to an inert aluminum oxide surface may
seem like a somewhat quixotic aim, but it has enormous potential in
using the high surface area productively (Chen et al., 2010; Javid et al.,
2006; Lee et al., 2002; Mateo et al., 2011; Milka et al., 2000; Mutalib et
al., 2009; Szczepanski et al., 2006; Wang et al., 2011). Surface
modification allows fine-tuning of the basic properties of alumina, e.g.
hydrophobicity, reflectivity and surface charge, but also membrane
selectivity, antifouling resistance and the recruitment of biomolecules
in specific and potentially high-throughput assays. Examples are
given in Table 3.
Aluminum oxide surfaces can be modified in several ways, which
are usually based on polymer adsorption or monolayer formation The
adsorption of poly-L-lysine to PAO results in positively charged surfaces that can bind DNA fragments by electrostatic interactions. This
has been exploited for the fabrication of a FRET based sensor with
single pore resolution (Masumoto et al., 2004). Similarly, the adsorption of poly(diallyldimethylammonium chloride) can facilitate gold
nanoparticle binding (Ko and Tsukruk, 2008). In situ polymer
formation on PAO has been demonstrated by plasma polymerization
(Losic et al., 2008). This yields an amine-terminated surface, but also
reduces the effective pore diameter.
The advantages of monolayer formation are that it is usually based
on self-assembly, resulting in full and uniform surface coverage, and
that it does not notably change the structural properties of the
substrate. Several classes of compounds are known to form monolayers on aluminum oxide, including carboxylic acids, organosilanes
and phosphonic acids (Fig. 5). The adsorption of n-alkanoic acids on
PAO is possible, but as this adsorption process is reversible, the
resulting monolayers were found to be unstable under aqueous
conditions (Chang and Suen, 2006). Most reports, however, deal with
organosilanes. This is remarkable, since the packing density and
stability of organosilane monolayers is known to be limited on alumina in comparison to other substrates. (Liakos et al., 2004; Oberg et
al., 2001). The deposition of silica prior to monolayer formation is
suggested to improve monolayer stability, but complicates the modification procedure (Szczepanski et al., 2006). Phosphonic acids, on the
other hand, do form stable monolayers on alumina (e.g. Liakos et al.,
2004; Yildirim et al., 2010), but there is only one example of the
adsorption of these compounds onto PAO (Koutsioubas et al., 2008),
possibly due to the limited commercial availability of functional
phosphonic acids. Finally, it was recently shown that chemisorption of
terminal alkynes onto PAO also results in the formation of stable
monolayers (ter Maat et al., 2011).
Modification of PAO with organosilanes has been applied to
change the surface charge (Chen et al., 2010), to create a superhydrophobic material (Park et al., 2010) and to reduce membrane
fouling (Popat et al., 2004; Yeu et al., 2009). In a more advanced study,
a preformed monolayer is used as the initiator for the controlled
growth of polymer brushes. Subsequent functionalization with a
nitriloacetate-copper complex results in a membrane that can be used
for affinity separation of histidine-tagged proteins (Sun et al., 2006).
An interesting development is the layered surface modification reported by Jani et al. (2009, 2010), in which anodization and organosilane monolayer formation are repeated. This process allows one
to further tune the transport properties of the membrane.
By coupling (functionalized) inorganic nanoparticles to the PAO
surface, new properties may also be introduced to the substrate. This
coupling can be facilitated by modification of the PAO with a polymer
or organic monolayer (Ko and Tsukruk, 2008), but can also be performed directly onto the PAO. (Thormann et al., 2007). Coupling of
nanoparticles to the PAO is often performed with the aim of Raman
enhancement (see Section 6), but also for the introduction of fluorogenic detection reagents. A modified PAO membrane was created by
covalently coupling fluorophore-loaded silica nanoparticles to the
PAO (Fig. 6). As the fluorophore (pHrodo, Invitrogen) displays
pH-dependent fluorescence, the resulting surfaces can be used for
screening of strains of acid-producing bacteria.
An exciting and commercially viable set of applications for functionalized surfaces is the recruitment of biomolecules for specific
assays — potentially massively parallel with a large number of binding
sites (e.g. immobilized antibodies or oligonucleotides) for different
targets (Wu et al., 2004). An elegant example is the use of PAO to
create silica nanotubes that are able to display functional antibodies
capable of distinguishing between drug enantiomers. Such a system
can selectively bind one enantiomer. An extension of this idea was to
reduce the affinity of the antibody, without removing selectivity, using
DMSO as a co-solvent. This allows a PAO surface to be used as a
chromatography system — effectively billions of nano-chromatography
columns in parallel, for the separation of high value biomolecules (Lee
et al., 2002). Nucleic acid microarrays, displaying oligonucleotides for
mRNA or the capture of single nucleotide polymorphisms (generally
amplified as PCR products) have also been developed (Wu et al., 2004).
These display rapid kinetics compared to planar arrays, with active
pumping of the target molecules through a 3D array of detector
molecules. This has been extended to peptide display arrays used in
C.J. Ingham et al. / Biotechnology Advances 30 (2012) 1089–1099
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Table 3
Biomolecules and biofunctional agents immobilized on PAO.
Molecule
Category
Direct PAO modification
Secondary
modification
Application
Reference
Alliinase
Phenol
isothiocyanate
Fluorescein
isothiocyanate
Gold
Polyethylene glycol
D N A
oligonucleotides
DNA
Enzyme
Fluorophore
Silane
Direct adsorption
Sugar-lectin
Bioreactor
Polychlorobiphenol sensor
Milka et al. (2000)
Wang et al. (2011)
Fluorophore
Silane
Mutalib et al. (2009)
Nanoparticles
Hydrophilic agent
Nucleic acids
Silane
Silane
Thiol/maleimide
POPa multilayered
functionalization
POP multilayered functionalization
Differential dye perfusion
Gene expression microarray
Nucleic acids
Poly-L-lysine
Gene expression microarray
Peptides
Protein
Protein
Carbohydrate
Nanoparticles
Polymerized
n-heptylamine
Hydrophilic agent
Proteins
Thiol/maleimide
Silica
Fluoro-organic acid
Unknown
Alkene
Kinase assay
Chiral drug separation
POP
Multivalent carbohydrate array
Detection microbial culture
POP
Matsumoto et al.
(2004)
Hilhorst et al. (2009)
Lee et al. (2002)
Cheow et al. (2007)
Pera et al. (‐)
See Fig. 5.
Losic et al. (2008)
Antifouling during filtration
Nitrilotriacetate-Cu2+
Popat et al. (2004)
Sun et al. (2006)
n-Alkanoic acids
Filtration
Contrast enhancement
POP protein adsorption
Thormann et al. (2007)
Durtshi et al. (2005b)
Chang and Suen (2006)
Silanation
Nucleic acid purification
Chang et al. (2008)
Kinase substrates
Antibodies
Antibodies (IgG)
Glycodendrimers
Dye loaded silica
Various via amine
Polyethylene glycol
BSA and myoglobin
Tetraether films
Nickel
Bovine serum
albumin
Ion exchange
polymers
a
Lipids
Nanoparticles
Protein
Silane
Poly(2-hydroxyethyl
methacrylate)
Direct lipid adsorption
Silane-aldehyde
Mutalib et al. (2010)
Mutalib et al. (2010)
Wu et al. (2004)
POP, proof of principle.
protein kinase assays (Hilhorst et al., 2009) and in multivalent
carbohydrate arrays (Pera et al., 2010). One of the advantages of PAO
arrays is that information on binding kinetics (e.g. kinase interacting
with a peptide substrate and how kinase inhibitors affect such an
interaction) can be obtained, something that is potentially valuable to
drug screening operations (Vivanco et al., 2010).
Moreover, surface functionalization may also allow the addition of
cell-specific detection reagents and cell-capture agents. This may
allow the recruitment, culture and detection of specific types of
bacteria. These types of assays are likely to have further roles in the
screening for microorganisms for industrial applications, but also in
the detection of infections in medical diagnostics.
6. Physical detection technologies
configuration’). This allows the monitoring of exceptionally subtle
changes in the refractive index due to adsorption of e.g. molecules or
proteins inside the pores. (Lau et al., 2004). This sensitivity can be
attributed to the well-ordered pore arrays (resulting in distinct
waveguide modes) and to the high surface area of the PAO. Such
composites of PAO/Al have proven competitive with surface plasmon
resonance (SPR) in terms of sensitivity (Yamaguchi et al., 2009). What
is more, it is also possible to extract spectral information on the
adsorbed compounds from these measurements (Hotta et al., 2010).
An alternative waveguide-based method using PAO was recently
shown to detect small concentrations of pesticide (Trivinho-Strixino
et al., 2010).
Thin PAO substrates have also been used as surface plasmon
resonance (SPR) sensors, using the same Kretschmann configuration
As described in Sections 3 and 4, PAO has a low background for
fluorescence measurements, which can be advantageous for cell
imaging studies. However, there are other imaging technologies that
are actively enhanced by PAO or by structures templated by PAO.
Sensitive optical detection is possible on nanoporous substrates. One
method is to apply the PAO as an optical waveguide, for example by
stacking a metal-supported PAO substrate to a prism (‘Kretschmann
Fig. 5. Surface modification of PAO. Examples of monolayer types that can be formed on
PAO, resulting from the adsorption of: A. Carboxylic acids, B. phosphonic acids, and
C. organosilanes.
Fig. 6. PAO cuture chip with intrinsic nanodetection reagent. A. Scanning electron
microscopy of PAO with up to 200 nm pores and customizable spherical 80 nm
nanoparticles loaded with pH sensitive reporter dye. The particles have been colored
blue-green to increase their visibility in this image. B. Coupling chemistry used to attach
nanoparticles to the PAO functionalized (F) with an alkyne surface chemistry.
C. Increase in fluorescence of nanoparticle (N) by decreasing pH.
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C.J. Ingham et al. / Biotechnology Advances 30 (2012) 1089–1099
(Koutsioubas et al., 2008). Similar to optical waveguides, SPR is also
used to monitor changes in the refractive index due to adsorption.
Comparison with planar surfaces showed improved sensitivity of the
PAO due to the increased surface area. This technique allowed
monitoring of the formation of octadecylphosphonic acid monolayers
on PAO (Koutsioubas et al., 2008). In an alternative approach, the
ordered structure of PAO was used as a template for the condensation
of nanocrystalline gold, which allowed the monitoring of antibody–
antigen interactions with localized SPR (Kim et al., 2008).
Raman spectroscopy is a detection method that is very suitable for
aqueous solutions. PAO is highly compatible with this technique, as it
has a low background in the visible and near-infrared range. In
addition, it can be used as a substrate for surface enhanced Raman
spectroscopy (SERS), which is an exceptionally sensitive, label-less
and potentially real-time variant (Moskovits et al. 2011). The
surface-enhancement effect is induced by strongly localized electromagnetic fields (‘hot spots’) that can be obtained by excitation of
nanostructured metals. Therefore, early studies used PAO as a
sacrificial template to form ordered metal nanostructures (Schierhorn
et al., 2006; Wang et al., 2006), In addition, SERS-active substrates
have been obtained by adsorption of silver and gold nanoparticles
onto PAO (Ji et al., 2009; Ko and Tsukruk, 2008; Ko et al., 2009; Lu et
al., 2009). These surfaces show increased sensitivity compared to
planar surfaces due to the higher surface area, but also due to the
optical waveguide effect. The latter was estimated to give rise to an
additional enhancement of about three orders of magnitude (Ko et al.,
2009), and corresponding substrates were used to detect concentrations of explosives to a sensitivity of one part in 10 12 (Ko et al., 2009).
Other approaches exploit the specific nanostructural features of
PAO to create SERS substrates. The highly ordered and organized PAO
was lined with a metal to obtain a surface with homogeneous SERS
activity, which is hard to obtain by nanoparticle-based methods (Choi
et al., 2010). Gold deposition within the nanopores, combined with
fine-tuning of the PAO thickness, has been used to create sensitive
arrays (Choi et al., 2010). Sputtering a well-ordered PAO substrate
with silver can result in the formation of hemispherical silver nanocaps on the outer surface. It was shown that the distance between the
individual nanocaps is able to be fine-tuned, which makes these
surfaces excellent SERS substrates (Qiu et al., 2009). In addition to
homogeneous SERS activity, the advantages of these methods are
their simplicity and scalability.
In addition to optical methods, there are also PAO-based detection
methods that rely on electrical conductance through the nanopores.
Because of the large surface to volume ratio of PAO, the ionic conductance through the pores is sensitive to the interaction of ions with
the walls. An electrical biomolecule sensor has been made, by
depositing gold as electrodes on both faces of the PAO. Binding of
DNA or RNA to a complementary nucleic acid sequence (or a neutral
nucleic acid analog) immobilized in the pores leads to a decrease in
ionic flux through the pores (Wang and Smirnov, 2009). The effect is
far larger in 20 nm pores compared to larger diameters. This method
has been used to detect single nucleotide polymorphisms, while a
related method was capable of assaying the effect of an anti-cancer
drug on mammalian cells arrayed in nanowells on PAO (Yu et al.,
2009).
PAO supports also show advantages for mass spectroscopy. Typically, conventional matrix-assisted laser desorption/ionization mass
spectroscopy (MALDI-MS) uses an organic matrix to transfer the
energy for desorption to the target biomolecules. MALDI-MS has been
developed into a key method for biomolecule analysis, but it has
some shortcomings that relate to the use of a matrix (e.g. significant
background in the low mass range). This has led to the development
of matrix-free LDI methods on nanotextured surfaces, including PAO.
It was found that effective desorption/ionization requires coating the
PAO with a metal (Okuno et al., 2005). Other parameters, such as the
porosity and the thickness of the PAO and metal coating, also
influence the signal intensity (Nayak and Knapp, 2007; Wada et al.,
2007). Absorption of the laser irradiation by the thin metal coating on
the thermally isolating PAO results in high local temperatures and
metal melting (Wada et al., 2007). Energy transfer to the analyte then
results in its desorption/ionization. PAO-based LDI has been shown to
give comparable performance to MALDI, but with lower backgrounds
in the low mass range. (Nayak and Knapp, 2007). In addition, the
samples are also compatible with desorption electrospray ionization
(DESI), a complementary ionization mode (Nayak et al., 2008).
In summary, the enhancement of a number of imaging and
detection technologies due to the PAO structure offers the opportunity for imaging methods to enter common usage, some of which may
otherwise be too specialized or limited to enter the market place.
Techniques that are already finding a place in routine diagnostics,
such as Maldi-TOF, may also benefit from sample carriers that include
PAO in their manufacture.
7. Indirect uses — PAO as the basis for fabricating
other nanostructures
The structure of PAO and the physical and chemical properties of
aluminum oxide are qualities that can be separated. PAO is capable of
acting as a structural template for other nanofabrication processes.
Broadly there are three possibilities: (a) an inverse structure is made
using PAO as a scaffold — effectively a nanowire or nanotube; (b) a
copy or at least a similar structure of the PAO structure is made
in another material; (c) the pores are used to create another structure that is neither a copy nor the complement of PAO, such as a
nanoparticle.
Inverse structures can be fabricated from polymers, metals or
semiconductors. The result is an array of nanowires or nanotubes
contained within the PAO pores or liberated as free structures. The
literature on the use of PAO as a scaffold is extensive, and several
dedicated reviews on this subject are available (e.g. Al-Kaysi et al.,
2009; Sarkar et al. 2007). Therefore, we only present a selection of
examples. One application of nanowires is the culture of mammalian
cells (Grimm et al., 2010). Nickel wires grown in PAO by electrodeposition can be aligned in a magnetic field to create surfaces for the
culture of mammalian cells. Externally imposed magnetic fields can be
used to exert precise forces on the cells (sometimes after internalization of a nanowire). This allows creation of unique textures and
patterns (coupled with surface modification (Johansson et al., 2010)).
Biodegradable scaffolds for cell and tissue engineering can also be
produced using PAO as a template. For example, poly(ε-caprolactone)
supports for stem cell research, neuronal cell regeneration and bone
tissue engineering have been created by extrusion of the polymer
through PAO. Sodium hydroxide is then used to destroy the PAO and
liberate arrays of nanowires with an average diameter of 200 nm
(Bechara et al. 2010, Porter et al. 2009). These permit the growth and
development of cell types that are difficult to achieve on other
surfaces. An interesting aspect of this method is the possibility to
incorporate active biomolecules into such a nanowire array; for
example agents designed to promote the differentiation of particular
cell lines.
Silica nanotubes can be fabricated within PAO pores then individually released by reactive ion etching to create silica nano test
tubes (Buyukserin and Martin, 2010). The etching process can also
close the nano test tubes to create capsules. The potential for surface
functionalization (e.g. antibody coating as a targeting mechanism),
low levels of cytotoxicity and rapid dispersal make these attractive as
a drug delivery system.
At least partial copies of the well structure of PAO are manufacturable using the alumina as a mask to selectively etch another material,
generally resulting in an array of nanowells that reflects the structure of
the mask (Kang et al., 2005). The PAO mask is simply placed on top of a
planar piece the material to be etched (e.g. glass, carbon, polymers,
C.J. Ingham et al. / Biotechnology Advances 30 (2012) 1089–1099
metals) and argon plasma used to selectively remove material to create
nanowells with a similar pattern to the mask. Porous silicon nanowell
arrays have been made in this way and applied to matrix-free laser
desorption mass spectroscopy of proteins and other biomolecules
(Gulbakan et al., 2010). Light emitting diodes have even been created in
semiconductors textured with nanowells using a PAO template (Soh et
al., 2010). This process can be thought of as a simple method of
fabricating textured nanosurfaces. However, it has also been pointed out
(Kang et al., 2005) that when combined with nanospheres that may vary
in surface properties, and which occupy individual nanowells, the
nanowell system can store chemical information that can be read out by
confocal microscopy or an array of optical detectors.
By evaporating aluminum onto PAO and melting of the metal,
inverse aluminum structures have been obtained. However, the
diameter of the obtained aluminum features was bigger than that of
the original PAO template, which was attributed to the penetration of
the aluminum into Al2O3 layer at the surface. This metal:metal oxide
composite confers improved mechanical properties, which allows the
templated structure to be used as a mold for nanoimprint lithography
(Lee et al., 2005). The PAO itself can also be used for imprinting
purposes. Hot embossing, where a (supported) PAO template is pressed
onto a polymer film at elevated temperature, has been reported for
polystyrene and cyclic olefin copolymer films (Koponen et al., 2007; Lee
et al., 2008). This resulted in inversion of the PAO pattern into the
polymer, yielding an array of polymer nanopillars. Similar structures
were obtained using UV nanoimprint lithograpy, in which a PAO template is pressed onto a polymer precursor, which is subsequently cured
with UV light to produce the nanostructured polymer (Bessonov et al.,
2010). Finally, injection molding of polyvinyl chloride into PAO
templates has been demonstrated, yielding a patterned polymer with
shallow features (Koponen et al., 2007).
Other types of nanoparticles can be created using PAO, as with nano
test tubes with applications in drug delivery. Uniform, nearly perfectly
spherical polymer nanoparticles can be created by using PAO to emulsify
monomers, which are photopolymerized after passaging through the
membrane, the pore size being used to control the final size of the
nanoparticle (Yanagashita et al., 2009). Particle size was slightly larger
than the pore size and nearly linearly related to the pore diameter.
Biodegradable polymers can also be created from materials such as
chitosan (a polymer of N-acetylglucosamine and glucosamine). The
method of creation is to passage the monomers through PAO so a rapid
increase in pH is achieved, allowing highly controlled polymerization
(Guo et al., 2010). Small molecules can be incorporated into chitosan
during this process. Finally, particles related to quantum dots can be
grown within the nanopores of alumina (Zelenski et al., 1997).
1097
ture, it is likely that the biotechnology and nanotechnology applications are sufficient to drive more widespread commercial PAO
formats. For the material to be widely usable within biotechnology,
further decreases in cost and improvements in durability are critical to
enable PAO to be used disposables or scaled-up processes. Here, the
bottom line may be the cost of high purity aluminum.
Surface chemistry, to date, generally functionalizes PAO in limited
ways. Several chemistries exist but generally the same modification is
made across a piece of this material. Where different biomolecules are
recruited to different areas (e.g. in fabricating a microarray with
different biomolecules in different spots) then this is usually done by
printing and a secondary conjugation. In the future we may expect
higher resolution and more versatile surface chemistries.
Two trends in detection methods are supportive of multiplexed assays
on PAO. The first is related to the advantages that the material brings in
terms of optical imaging methods (SERS, optical wave guides). If these
sophisticated and powerful methods can be brought to the marketplace
then this opens up powerful types of reagent free imaging. Additionally,
advances in simple and cheap illumination methods, particularly LEDs
(Albeanu et al., 2008), and the emergence of new detection reagents
(nanoparticles) should allow fluorescence-based detection methods to
increase in power. Trends in imaging, such as low cost digital data capture,
USB microscopes, direct detection of cell autofluorescence using CCD
chips (London et al., 2010) can be expected to support wider usage. The
decrease in LED costs and cost-per-pixel of digital data capture supports
the increasing miniaturization of microbial culture with low cost and
point-of-testing and/or online read-out methods, a goal that has been
described as telemicrobiology (Scheid et al., 2007).
Within microbiology there is some competition between molecular
and culture based approaches. However, the failure of molecular
techniques to displace traditional culture methods indicates that both
approaches are necessary. One logical, but not entirely easy, step would
be assays that integrate elements from both technology streams. This
would allow a combination of phenotyping and genotyping, or
identification of a target organism combined with an analysis of whether
it is viable and what it is capable of (e.g. pathogenicity or carcogenicity).
This goal could be achieved on PAO systems by capturing organisms
based around their surface properties (e.g. an antibody) followed by
culture; or the reverse — culture then lysis and molecular analysis.
Materials can have a major impact on biotechnology. For example,
relatively simple and inexpensive plastics have transformed many
aspects including cell growth and high throughput screening through
disposables, such as the multiwell plate and the disposable Petri dish.
We are only just learning to leverage the potential of nanoporous
materials in biology, but it is our expectation that such materials,
including PAO, will have a significant impact too.
8. Future prospects
Diverse and inventive minds have seized upon the properties of
PAO and exploited this material in biotechnology applications as
wide-ranging as molecular separations, cell imaging, high throughput
screening, sensors of environmental hazards, drug delivery, tissue
engineering and single molecule imaging (Fig. 3). The structural
properties of PAO have been transferred to other materials, as inverse
structures (nanotubes and nanowires) or by replicating the pore
pattern by using the PAO as a shadow mask. Improvements in the
basic structure of PAO have resulted in more regular pores. Alterations
in the surface properties of PAO have been achieved by chemical
functionalization. Functionalization has allowed fine-tuning of basic
surface charge and hydrophobicity, but also the specific capture of
biomolecules, facilitating highly multiplexed assays.
Despite intense interest, we still do not completely understand the
self-ordering process that anodization brings to aluminum. Therefore,
it is necessary to investigate this phenomenon more completely,
which should, in turn, unlock further advances in controlling and
extending the range of PAO variants available. In terms of manufac-
Acknowledgments
This work was supported in part by the Nano4vitality and
RAAKPro programs and an unrestricted Spinoza award of the
Netherlands Organization for Scientific Research (WMdV).
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