Nanoscience and nanotechnology involve the study, imaging, measuring, modelling,
or manipulation of matter at the molecular and nanometre scale. The application
of nanotechnology and nanoscience to biological systems - known as bionanotechnology1
- holds the promise of solving many of today's challenges in healthcare.
Dramatic breakthroughs are expected in life sciences research that could contribute
to sensitive and early detection of a number of human diseases, such as cancer,
Alzheimer's disease, multiple sclerosis or rheumatoid arthritis.
Therapeutic fields such as drug delivery, tissue engineering, and drug discovery,
will also benefit greatly from advances in bionanotechnology. Although often
considered1 one of the key technologies of the 21st
century, bionanotechnology is still in a fairly embryonic state and much of
its potential is yet to be realised. Surface bionanotechnology - the science
and technology of understanding and precisely controlling and manipulating surface
biological properties at the molecular and nanometre scale - is one of most
exciting and potentially important areas of bionanotechnology.
The objective of Dr.
Mendes' research at the University
of Birmingham is to further develop the interdisciplinary surface bionanotechnology
field both on a fundamental level and towards biological and medical applications.
Mendes' research group aims to generate surface materials with biological
properties that are precisely controlled and manipulated at the molecular and
nanometre length scales by interfacing nanotechnology with biological systems.
This field of research is one of most exciting and potentially important areas
of bionanotechnology, but so far limited progress has been made partly due to
the complexity involved in the design and fabrication processes. In order to
tackle this ambitious challenge, we have been developing breakthrough methodologies
for engineering patterned2 and stimuli-responsive3
surfaces. Within the field of patterned biological surfaces, Dr.
Mendes' research group in collaboration with Machesky group (The Beatson
Institute for Cancer Research, UK) has developed4
a methodology to control the spatial immobilisation of cell-adhesive (fibronectin)
and non-cell-adhesive (bovine serum albumin - BSA) proteins in specific
micro-areas of a glass surface in order to gain valuable insights into how cells
explore their environment and form new adhesion contacts for motility and spreading.
Mendes used micro-contact printing (µCP) and mouse embryonic fibroblast
(MEF) cells for the study of both filopodia and filopodial/lamellipodial transitions.
µCP was employed to create fibronectin linear patterns with widths varying
from 10 µm to 2.5 µm and spacings between 10 µm and 0.5 µm
that were backfilled with BSA. Fibronectin regions provided an activating signal
and an adhesive surface, while denaturated BSA regions enabled us to examine
how cells interact with non-adhesive areas. MEF cells were able to spread on
these patterned surfaces, and for wider fibronectin widths and BSA gaps (5 µm
x 5 µm and 10 µm x 10 µm patterned surfaces) the orientation
of spreading was always in the direction of the striped pattern (Figure 1).
Images are single frames from a timelapse of a MEF spreading on 10 µm
By using these wider linear patterned surfaces, it was also possible to observe
that the size of lamellipodia was not dependant on the width of the stripe and
that lamellipodia, but not filopodia, require adhesion for persistent protrusion.
These studies also allowed us to learn more about the involvement of Arp2/3
complex in cell spreading, and in particular, that the localisation of Arp2/3
complex to filopodia is independent of adhesion. More recently, Dr.
Mendes has developed5 a protocol to create robust
and high resolution patterns of bacteria onto material surfaces, allowing arrays
of viable cells to be developed with controlled spatial structure for a variety
of experimental procedures including cell-to-cell communication studies.
Within the field of stimuli-responsive surfaces, Dr.
Mendes' research group has developed6 electro-active
surfaces that have been successfully employed to switch on functionalities in
situ, offering an unprecedented ability to manipulate the interactions of proteins
with surfaces. A new class of switchable biological surfaces - surface-confined
electro-switchable peptides - have been also generated7
that have the capacity to regulate biomolecular interactions in response to
an applied electrical potential.
This system is based upon the conformational switching of positively charged
oligolysine peptides that are tethered to a gold surface, such that bioactive
molecular moieties - biotin - incorporated on the end of the oligolysines
can be exposed (bio-active state) or concealed (bio-inactive state) on demand
(Figure 2), as a function of surface potential. The oligolysine peptides exhibit
protonated amino side chains at pH =7, providing the basis for the switching
"On" and "Off" of the biological activity on the surface.
For instance, upon application of a negative potential, the positively charged
molecular system experiences an electrostatic attraction to the surface, leading
to a mechanical molecular motion that shields the bioactive moiety (Figure 2).
In order to test the viability of the proposed switching mechanism we selected
an end functionalised biotinylated peptide as the biorecognition motif, four
lysine residues as the switching unit and a terminal cysteine for self-assembled
monolayer (SAM) formation on gold surfaces - biotin-Lys-Lys-Lys-Lys-Cys (Biotin-KKKKC).
Schematic representation of the switching of mixed TEGT-biotinylated
peptide SAMs between a bio-active and bio-inactive state. Depending
on the electrical potential applied, the peptide can expose or conceal
the biotin site and regulate its binding to NeutrAvidin.
In order to provide sufficient spatial freedom for each biotinylated peptide
on the surface, such that it can undergo conformational changes upon switching
without steric hindrance from neighbour molecules, gold surfaces were functionalised
with a two-component, mixed SAM of the biotin-KKKKC peptide and tri(ethylene
glycol)-terminated thiol (TEGT) (Figure 2).
Apart from ensuring sufficient spatial freedom for synergistic molecular reorientation
of the surface-bound biotinylated peptide, the short oligo(ethylene glycol)
groups prevent nonspecific interaction from the proteins. As a control, a two-component,
mixed SAM was also prepared using TEGT and a peptide without the biotin moiety
- KKKKC. The dynamics of switching the biological properties was studied by
observing the binding events between biotin and fluorescently labeled NeutrAvidin.
Fluorescence microscope images and SPR spectral data clearly revealed binding
at + 0.3 V, reduced binding at - 0.4 V, and intermediate binding in the
at open circuit (OC) conditions (no applied potential). Depending on the electrical
potential applied to the mixed SAMs, bioactive molecules incorporated onto the
SAM can be fully exposed for binding (+ 0.3 V, bio-active state) or concealed
(- 0.4 V, bio-inactive state) to the extent that binding affinity can
be reduced to over 90% of its bio-active state (Figure 3). Furthermore, reversibility
studies by SPR also demonstrated that the developed switchable surface allows
reversible control of biomolecular interactions.
SPR sensorgram traces showing the binding of NeutrAvidin to the Biotin-KKKKC:TEGT
mixed SAMs and KKKKC:TEGT mixed SAMs under OC conditions and an applied
positive (+ 0.3 V) and negative (- 0.4 V) potential.
This surface technology takes advantage of the unique dynamic properties of
surface-confined charged peptide linkers at the nanometer scale to induce On-Off
switching of specific biomolecular interactions, setting the stage for advances
in biological research, medicine, biotechnology, and bioengineering.
1. C. M. Niemeyer, C. A. Mirkin, Wiley-VCH Verlag GmbH &
Co. KGaA, Weinheim, 2004.
2. P. M. Mendes, C. L. Yeung, J. A. Preece, Nanoscale Research
Letters 2007, 2, 373.
3. P. M. Mendes, Chemical Society Reviews 2008, 37, 2512.
4. S. A. Johnston, J. P. Bramble, C. L. Yeung, P. M. Mendes,
L. M. Machesky, BMC Cell Biology 2008, 9, 65.
5. C. Costello, J.-U. Kreft, C. M. Thomas, P. M. Mendes, Nanoproteomics:
Methods and Protocols, S. Toms and R. Weil, eds., in Springer Protocols. Methods
in Molecular Biology, Humana Press. In Press.
6. P. M. Mendes, K. L. Christman, P. Parthasarathy, E. Schopf,
J. Ouyang, Y. Yang, J. A. Preece, H. D. Maynard, Y. Chen, J. F. Stoddart, Bioconjugate
Chemistry 2007, 18, 1919.
7. C. L. Yeung, P. Iqbal, M. Allan, M. Lashkor, J. A. Preece,
P. M. Mendes, Advanced Functional Materials, 2010, 20, 2657.
Copyright AZoNano.com, Dr. Paula Mendes (University of Birmingham)
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