Characterization and Fabrication Tools for Emerging Nanobionics

  • Fig, 1:  A) Source: . Highlights the effect of increasing the number of electrodes on the quality of vision received from the Bionic Eye Implant. B) Integration of nanoscale electrodes with single nerve cell.Fig, 1: A) Source: . Highlights the effect of increasing the number of electrodes on the quality of vision received from the Bionic Eye Implant. B) Integration of nanoscale electrodes with single nerve cell.
  • Fig, 1:  A) Source: . Highlights the effect of increasing the number of electrodes on the quality of vision received from the Bionic Eye Implant. B) Integration of nanoscale electrodes with single nerve cell.
  • Fig. 2: DPN as a nanofabrication tool for electroactive materials.  A) Schematic and AFM image of a single feature of a PEDOT:PSS within a patterned array. B) Line writing of the liquid oxidant ink spelling IPRI (for the Intelligent Polymer Research Institute).  Each line is <300 nm in width.  C) Optical microscopy images of a single platinum dot deposited on a strand of human hair. Both the reflectance image (left) and transmission image (right) are shown.
  • Fig. 3:  A) Shape memory effect of a polythiophene conducting polymer film under electrical control. Nanoindentations (circled) of 30-40 nm in diameter are formed in the polymer by indenting the surface with the AFM tip prior to electrical stimulation (top panel). The same nanoindentations can be ‘erased’ upon the application of -1 volt (middle panel) and subsequently restored by reversing the voltage to +1 (bottom panel). B) 3D –AFM height image showing the cyclic change in the expansion and contraction of a thin film conducting polymer during an applied AC potential.
  • Fig. 4:  A) Schematic showing fibronectin functionalized AFM tip interacting with a conducting polymer electrode doped with biopolymers. B) AFM force curves showing difference in adhesion force with no electrical stimulation and during the application of +400 mV. The extent of protein binding is given by the magnitude of peaks in the curve. C) Graph showing that adhesion reversibly increases and decreases with cycling of the potential. Adhesion force values (black dots) are measured during the application of a cyclic voltammogram (red curve, arrows show direction of scan).

The interfacing of technology and living cells in the nanoscale domain -nanobionics- is being enabled by scanning probe tools, including Bio-Atomic Force Microscopy and Dip Pen Nanolithography, providing a nanoscale insight into the cell-material interface and an unprecedented approach to nanofabrication.


Bionic devices restore human function by integrating electronic technology with the body. The cochlear implant has been an outstanding success in the restoration of nominal hearing in profoundly deaf patients. This success has come using an electrode array-the part of the device in functional contact with living tissue-of 22 or 24 millimetre-sized platinum electrodes in silicone rubber, and fabricated using decades old manufacturing processes. Characteristic of the structure and material composition of the implant are the design paradigms to be minimally invasive, and maximally inert. We expect future generations of bionic implants to engender a shift in these paradigms whereby the implant will be structured-chemically, physically, electroactively-at smaller and smaller scales in order to offer several new dimensions of integration with nervous tissue (fig. 1). Future implants will promote tissue integration, over inertia. This more intimate connection between man and machine will result in a new range of diverse bionic therapies-such as spinal cord repair, muscle regeneration, the bionic eye and improved cochlear implants. Fundamental to these advances are the enabling tools which help us to understand, and manipulate, the cell-material interface at the nanoscale.

Living cells are microscopic, yet their sensory range extends well into the nanodomain. The focal adhesions which cells use to attach to a material, or to other cells, are just 70 nm in diameter. Cellular behaviour can be influenced by nanoscale differences in the surface topography encountered. The differentiation of stem cells down a particular pathway can be influenced by the specific chemical functionality of their nanoenvironment in vitro. In order to understand cellular behaviour, our characterisation tools must be at least as sensitive as the cells themselves-our tools must probe at the nanoscale and molecular level.

Similarly, nanobionic structures must be assembled in such a way that control over the distribution of material composition is possible with nanometre resolution.

The ideal cochlear implant would consist of 30,000 electrodes-one to address each of the auditory neurons in the average human ear. Miniaturisation, however, cannot be achieved by a simple geometric shrinkage of conventional electrode designs. The choice of material must be informed by the scaling of relevant properties as electrode area is decreased. For example, as we shrink a gold or platinum electrode, the poor charge injection capability of noble metals becomes a limiting factor. On the other hand, the performance of other materials may actually increase with decreasing size.

We have recently focused on the development of organic conductors as materials with multifunctional capabilities as nanobionic electrodes. Organic conducting polymers (OCPs), for example, have a much larger charge injection capacity compared with noble metals and display faster electrochemical switching speeds at smaller dimensions. The incorporation of biomolecules as dopants, within the electrode itself enables the novel possibility of the controlled release of growth factors to attract neuronal growth cones, or the presentation of specific binding sites for cell attachment. The tuneable stiffness of OCPs mean these electrodes may be designed so as to integrate with the body without promoting a scar tissue response due to mechanical mismatch, as occurs with metal electrodes.
The development of the field of nanobionics is laced with a number of unique and stimulating challenges. In particular, we must, ultimately understand how cells feel and respond to their nanoscale environment and be able to design and fabricate nanostructured architectures which will promote intimate electrical contact while discouraging necrosis and scar tissue formation. We are adapting scanning probe microscopy to address each of these challenges-both as a tool for nanofabrication, and a tool for probing the cell-material interface with nanodimensional resolution.

Dip-pen Nanolithography as a Fabrication Tool for Nanobionics

Following its invention in 1986, atomic force microscopy rapidly evolved from its original purpose of visualization to encompass a wide range of characterisation capabilities, including the measurement of mechanical forces of adhesion and indentation, as well as local electric and magnetic properties. More recently, the capability of the AFM tip as a tool for fabrication has been realised, and a rapid evolution of different AFM lithography technologies is now in progress. Dip-pen nanolithography is an additive lithography technique which uses an atomic force miscroscope tip to "direct-write" functional materials. The key advantage of DPN is the excellent resolution achievable using a variety of different material ‘inks'. Many of the essential ingredients of prospective nanobionic devices can be printed by DPN including proteins, metals and conducting polymers. DPN also has the unique capability to ‘multiplex' several different inks at nanometer resolution within a subcellular area. We envisage the possibility of "writing", for example, both attractive and repulsive axon guide proteins at dimensions smaller than the axonal growth cone itself, or of writing nano-electrodes or nanocircuitry directly onto bionic materials.

Key to understanding the versatility of DPN is a knowledge of its two principle modes of ink transport: meniscus transport and liquid ink deposition. In the meniscus transport mode, the ink (usually a self-assembled monolayer forming molecule, such as an alkanethiol) is dried onto an AFM tip. When the tip is brought in contact with the substrate a water meniscus is formed between tip and surface due to capillary condensation. The ink molecules dissolve into this water meniscus and migrate to the surface. A chemical affinity between the ink and substrate (in this case, the thiol-gold bond) results in covalent attachment of the molecules. The liquid deposition method, on the other hand, uses a carrier solvent to assist ink transport to the surface via capillary action. Liquid ink transport extends the range of printable DPN inks to include metal nanoparticles, proteins and conducting polymer dispersions.

We have directed our efforts towards developing novel methods of using DPN to pattern electromaterials, such as conducting polymers and metals. Our goal is to build functional devices so as to take advantage of the favourable scaling of conducting polymer performance approaching the nanoscale.

The conducting polymer PEDOT:PSS has become one of the most important conducting polymers and is commercially available from several companies as an aqueous dispersion suitable for many processing techniques such as ink-jet printing, spray coating, spin coating etc. Beginning with one such commercially available solution, we used an alcohol dilution step to render the dispersion printable by DPN [1] (fig. 2A). Exploiting the versatility of liquid ink DPN, we found we could print the diluted ink on a variety of hard and soft, flexible substrates including silicon, gold, indium tin oxide (ITO), silicone gum and polyethylene terephthalate (PET) at submicrometer resolution. Following on from this work, we developed a second method of DPN printing PEDOT:PSS, this time exploiting some tricks of ink development borrowed from the ink-jet printing industry (namely, incorporation of additives to modify ink viscosity and surface tension) [2]. Glycerol was chosen as it has the concomitant effect of increasing the conductivity of PEDOT:PSS via a secondary doping mechanism and increasing viscosity. We found that, by synthesising the polymer in-house so as to control the ratio of PEDOT to PSS, we could create features with 150 nm diameter.

A further method of DPN printing conducting polymers aimed to marry the superb resolution achievable by DPN with the rapid advances currently being made in the vapour phase synthesis of conducing polymers [3]. We formulated a liquid ink based on the oxidant iron (III) tosylate stabilised by a block copolymer surfactant. This liquid oxidant was printable in attolitre aliquots where each printed feature could serve as a discrete nanoreactor for the in-situ synthesis of conducting polymer when exposed to a monomer vapour (fig. 2B). Importantly, this methodology could generate line patterns with widths down to 250 nm.

More recently, we have developed a method to print noble metals (both gold and platinum) at nanometer resolution on various flexible substrates. This method, again using a liquid based ink, utilises a metal precursor ink which is patterned onto a substrate and subsequently reduced in-situ to zero-valent metal. The ink is versatile enough, and the reduction step mild enough, that we can print and reduce platinum in situ onto a strand of human hair without damaging the hair (fig. 2C).

The utility of a single-pen DPN system is probably limited to niche applications requiring the localised functionalization of surfaces and micro- or nano-structures. Upscaling of the technology has been achieved using linear arrays of cantilevers (to print a dozen simultaneous patterns) and, more recently, by the invention of polymer pen lithography whereby millions of nanoscale features can be simultaneously printed over centimetre areas. These developments highlight the potential of scanning probe based lithography to develop beyond a tool of lab research, and into a tool of nanofabrication.

The inception of AFM represents a landmark event along the timeline of nanotechnology but more recently over the last decade the use of AFM in the biological sciences has increased rapidly due to its ability to operate under aqueous physiological conditions, measure across different length-scales ranging from single living cells through to individual proteins, and easily integrate with optical/fluorescence microscopy and electrical techniques to enable simultaneous acquisition of a wide range of physical, chemical and electrical information. The advent of a broader range of available commercial AFM systems and different modes of AFM provides new opportunities for advanced research and experimental flexibility. For example, AFM systems and designs (e.g. petri dish holders and heaters) that integrate well with optical techniques and facilitate live cells studies, including electrochemical cell configurations that enable observations of cellular dynamics as a function of electrical stimulation, represent exciting developments in this area [4]. These attributes of Bio-AFM fundamentally makes for an ideal characterization technique when one merges nanotechnology with biology in the field of Nanobionics.

From a material perspective, characterizing the nanoscale properties of OCPs as a function of electrical stimulation in biologically relevant environments is accomplished by implementing electrochemical apparatus within AFM [5]. Within such a setup, for example Electrochemical-AFM (EC_AFM), we have been able to observe reversible, dynamic, nanoscale changes in topography, local nanoscale materials stiffness and actuating properties of OCP in phosphate buffer saline and cell culture media. For example, nanoscale patterns written into the surface of a polythiophene OCP can change shape to the extent that they disappear in response to an applied voltage [6]. The nanopatterns can be restored to their original shape by reversing the polarity of the applied voltage (fig. 3A). Movement of ions in and out of the polymer, which exerts energy to change, or hide, the polymer structure of the nanoscale patterns, is believed to be responsible for this unique shape memory effect under electrical control. The electrical stimulation of thin film OCPs similarly reveals that the polymers expand and contract, or actuate, due to the reversible exchange of ions in the electrolyte [7]. This is visualized as a reversible height change in the 3D-AFM images (fig. 3B). While the actuation process is well understood for macro-sized films, EC-AFM provides the capability of measuring actuation processes with movements down to ~1 nm. The ability to observe dynamic changes in movement, structure and materials properties with high resolution paves the way for advances in new and interesting applications in nanobionics, particularly in this case where conducting polymers are utilized as MEMS devices, actuators and other micromachines for physically manipulating biological entities on the nanoscale.

Electrical stimulation is capable of controlling cell interactions, or acting as a "molecular switch" to turn on and off cell adhesion or differentiation. At present, electrically controlling the interactions of proteins such as fibronectin, a well-known mediator of cell adhesion, is a key strategy. For example, the electrically dependent conformation of adsorbed fibronectin is believed to be the underlying mechanism by which cell adhesion and migration are controlled on the OCP platform [8]. Deducing the "switching" mechanisms of single protein interactions is challenging and we are only just beginning to gain insight into the effect of electrical stimulation. Bio-AFM presents exciting opportunities - providing molecular level insights into the cell-electromaterial interface. Our group is currently using force spectroscopy to understand the interactions between single fibronectin proteins and OCPs doped with different biopolymers such as hyaluronic acid and chondroitin sulfate [9]. The principle behind this involves the use of a protein functionalized AFM tip that is brought into contact with the polymer and then withdrawn the protein-surface interaction forces acting on the tip are detected as it is withdrawn (fig. 4A). The implementation of EC-AFM further enables the measurements to be performed as a function of electrical stimulation. Without electrical stimulation, a single protein is able to bind via a specific set of peptide domains (i.e. heparin domains) to anionic groups of the biopolymers embedded within the OCP. During the application of a positive bias, the strength of protein binding dramatically increases by an order of magnitude (fig. 4B). This change in the protein interaction from weak to strong binding is fully reversible upon applying a cyclic potential (fig. 4C). These findings provide unprecedented insights by resolving the biophysics of the protein interaction at the single molecule level under electrical control. This is made possible by the direct measurement of biomolecular recognition at an electrode surface, which will be of significant interest in the areas of organic bioelectronics, electrical-based biosensing and implantable electrodes (e.g. cochlear implant), particularly as researchers endeavour to fabricate organic electrodes that make better electrical ‘connections' to proteins, living cells and tissues.


The emergence of nanobionics promises to enable dramatic improvements in existing medical bionic devices (such as the Cochlear implant) and delivery of next generation devices such as the bionic eye and implantable devices for epilepsy detection and control. To realise these possibilities several challenges need to be addressed. A better understanding of controllably and precisely depositing nanoparticulates, for example OCP nanoparticles, onto a range of different surface chemistries via DPN is required. The reliance on solely fundamental forces to drive the deposition process from the DPN tip may not be sufficient, and approaches that provide energy input (e.g. electro-assisted deposition) will be necessary. A patterned feature may also consist of only a few nanoparticles, therefore the issue of stability on the surface, particularly in aqueous environments, is a consideration. Integration of the DPN process into a fabrication stream for devices that span the micro- to nanometer range will also be important. In order to then utilize nanoscale electrodes based on OCP, we need a much better understanding of the cellular - material interface. For example, how do we implement these materials so that they make a better electrical ‘connection' to the living cell or tissues? Or how can we harness the dynamic, electromaterial surface properties to control cell interactions? These will require an ability to guide cell growth to the electrodes, enhance cell-electrode contact and adhesion, tailor surface chemistry for biomolecular/cellular recognition of the electrode surface, and then ultimately use electrical stimulation via the electrode to control the cell interactions. It is techniques like Bio-AFM that may provide the clues to many of these interactions at the molecular level.

This work has been supported by the Australian Research Council under the Australian Research Fellowship and DP110104359 (Dr Michael Higgins) and ARC Federation Fellowship of Prof. Gordon Wallace. We also greatly acknowledge the Australian National Fabrication Facility (ANFF) for providing Atomic Force Microscopy instrumentation.

[1] Nakashima, H.; Higgins, M. J.; O'Connell, C.; Torimitsu, K.; Wallace, G. G. Liquid deposition patterning of conducting polymer ink onto hard and soft flexible substrates via dip-pen nanolithography. Langmuir  2012, 28, 804-11.
[2] O'Connell, C. D.; Higgins, M. J.; Nakashima, H.; Moulton, S. E.; Wallace, G. G. Vapor Phase Polymerization of EDOT from Submicrometer Scale Oxidant Patterned by Dip-Pen Nanolithography. Langmuir  2012, 28, 9953-60.
[3] Wagner M.; O'Connell C.D.; Harman, D.; Ivaska, A; Higgins, M.J.; Wallace, G.G. Development and optimization of PEDOT:PSS based ink for printing of nanoarrays by Dip-Pen Nanolithography (submitted)
[4] For example:
[5] Higgins, M. J., Gelmi, A., Wallace, G. G.. Electrochemical-AFM for Understanding the Cellular-Electromaterial Interface. Imaging & Microscopy, 11(2):40. (2009)
[6] Higgins, M. J., Grosse, W., Wagner, K., Molino, P. J. and Wallace, G. G.. Reversible Shape Memory of Nanoscale Deformations in Inherently Conducting Polymers without Reprogramming. The Journal of Physical Chemistry B, 115: 3371. (2011)
[7] Higgins, M. J., McGovern S., Wallace, G. G.. Visualizing Dynamic Actuation of Ultrathin Polypyrrole Films. Langmuir, 25: 3627. (2009)
[8] Higgins, M. J., Molino, P., Yue, Z. and Wallace, G. G. Organic Conducting Polymer - Protein Interactions. Chemistry of Materials. 24: 828, (2012)
[9] Gelmi, A., Higgins, M. J., Wallace, G. G. Resolving sub-molecular binding and electrical switching mechanisms of single proteins at electroactive conducting polymers. Small. (accepted) (2012)

More information:
Organic Bionics, Wiley-VCH, Weinheim, ISBN: 978-3-527-32882-6
• A DVD on Bio-AFM for understanding biomolecular interactions is also available
(please email

Michael J Higgins
(corresponding author)
Cathal O'Connell
Amy Gelmi
Simon E Moulton
Gordon G Wallace

ARC Centre of Excellence for Electromaterials Science (ACES)
Intelligent Polymer Research Institute (IPRI)
AIIM Facility, Innovation Campus, University of Wollongong,
NSW, 2522, Australia


University of Wollongong, Bionics Group
Fairy Meadow
NSW 2519 Wollongong

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