Polymeric coatings which allow the effective control of biointerfacial interactions and cellular responses are of
increasing interest in a range of biomedical applications in vitro and in vivo such as cell culture tools, biosensors and
implantable medical devices. A variety of coating strategies have been developed to gain control over cell-surface
interactions but many of them are limited with respect to their function and transferability between different substrate
materials. Here, our aim was to establish an easily transferable coating that reduces non-specific cell-surface interactions
to a minimum while at the same time presenting functional groups which allow for the subsequent immobilisation of
bioactive signals. To achieve this, we have applied an allylamine plasma polymer coating followed by the covalent
immobilisation of a macro-initiator providing iniferter functional groups. Subsequent controlled free radical graft
polymerisation using the monomers acrylamide and acrylic acid in different molar ratios resulted in highly uniform
polymer coatings. Non-specific cell attachment was significantly reduced on coatings representing molar ratios of less
than 10% acylic acid. At the same time, we have demonstrated the suitability of these coatings for the subsequent
covalent binding of bioactive compounds carrying amine functional groups using the label 2,2,2-trifluoroethylamine.
Successful surface modifications were confirmed by X-ray photoelectron spectroscopy (XPS) and profilometry. The
cellular response was evaluated using HeLa cell attachment experiments for up to 24 hours. We expect that the coating
platform established in this study will be translated into a number of biomedical applications, including applications in
implantable devices and regenerative medicine.
Materials assisting with the efforts of cell isolation are attractive for numerous biomedical applications including tissue
engineering and cell therapy. Here, we have developed surface modification methods on microparticles for the purposes
of advanced cell separation. Iron oxide nanoparticles were incorporated into 200 ìm polystyrene microparticles for
separation of particle-bound cells from non-bound cells in suspension by means of a permanent magnet. The polystyrene
microparticles were further encoded with fluorescent quantum dots (QD) as identification tags to distinguish between
specific microparticles in a mixture. Cluster of differentiation (CD) antibodies were displayed on the surface of the
microparticles through direct adsorption and various methods of covalent attachment. In addition, a protein A coating
was used to orientate the antibodies on the microparticle surface and to maximise accessibility of the antigen-binding
sites. Microparticles which carried CD antibodies via covalent attachment showed greater cell attachment over those
modifications that were only adsorbed to the surface through weak electrostatic interactions. Greatest extent of cell
attachment was observed on microparticles modified with protein A - CD antibody conjugates. B and T lymphocytes
were successfully isolated from a mixed population using two types of microparticles displaying B and T cell specific CD
antibodies, respectively. Our approach will find application in preparative cell separation from tissue isolates and for
microcarrier-based cell expansion.
Gradient surfaces have become invaluable tools for the high-throughput characterisation of biomolecule- and cellmaterial
surface interactions as they allow for the screening and optimisation of surface parameters such as surface
chemistry, topography and ligand density in a single experiment. Here, we have generated surface chemistry gradients on
oxidised porous silicon (pSi) substrates using silane functionalisation. In these studies, pSi films with a pore size of 15-
30 nm and a layer thickness of around 1.7 ìm were utilised. The manufacture of gradient surface chemistries of silanes
was performed using a simple dip coating method, whereby an increasing incubation time of the substrate in a solution
of the silane led to increasing surface coverage of the silane. In this work, the hydrophobic n-octadecyldimethyl
chlorosilane (ODCS) and pentafluorophenyldimethyl chlorosilane (PFPS) were used since they were expected to
produce significant changes in wettability upon attachment. Chemical gradients were characterised using infrared (IR)
spectroscopy, X-ray photoelectron spectroscopy (XPS) and sessile drop water contact angle measurements. In addition,
the surface chemistry of the gradient was mapped using synchrotron IR microscopy. The ODCS gradient displayed
sessile drop water contact angles ranging from 12° to 71°, confirming the successful formation of a gradient. IR
microscopy and an XPS line scan confirmed the formation of a chemical gradient on the porous substrate. Furthermore,
the chemical gradients produced can be used for the high-throughput in vitro screening of protein and cell-surface
interactions, leading to the definition of surface chemistry on nanostructured silicon which will afford improved control
of biointerfacial interactions.
The ability to evaluate and control the cellular response to substrate materials is the key to a wide range of biomedical
applications ranging from diagnostic tools to regenerative medicine. Gradient surfaces provide a simple and fast method
for investigating optimal surface conditions for cellular responses such as attachment and growth. By using two
orthogonal gradients on the same substrate, a large space of possible combinations can be screened simultaneously. Here,
we have investigated the combination of a porous silicon (pSi) based topography gradient with a plasma polymer based
thickness gradient. pSi was laterally anodised on a 1.5 × 2.5cm2 silicon surface using hydrofluoric acid to form a pore
size gradient along a single direction. The resulting pSi was characterised by SEM and AFM and pore sizes ranging from
macro to mesoporous were found along the surface. Plasma polymerisation was used to form a thickness gradient
orthogonal to the porous silicon gradient. Here, allylamine was chosen as the monomer and a mask placed over the
substrate was used to achieve the thickness gradient. The analysis of this chemistry based gradient was carried out using
profilometry and XPS. It is expected that orthogonal gradient substrates will be used increasingly for the in vitro
screening of materials used in biomedical applications.
Transfection cell microarrays (TCMs) are a high-throughput, miniaturised cell-culture system utilising reverse
transfection, in which cells are seeded onto a DNA array resulting in localised regions of transfected cells. TCMs are
useful for the analysis of gene expression, and can be used to identify genes involved in many cellular processes. This is
of significant interest in fields such as tissue engineering, diagnostic screening, and drug testing[1, 2].
Low transfection efficiency has so far limited the application and utility of this technique. Recently, the
transfection efficiency of TCMs was improved by an application of a high voltage for a short period of time to the DNA
array resulting in the electroporation of cells attached to the surface[3, 4]. Furthermore, application of a low voltage for a longer period of time to the DNA array was shown to improve the transfection efficiency by stimulating the desorption
of attached DNA, increasing the concentration of DNA available for cellular uptake. In the present study, the
optimisation of the uptake of adsorbed DNA vectors by adherent cells, utilising a voltage bias without compromising cell
viability was investigated. This was achieved by depositing negatively charged DNA plasmids onto a positively charged
allylamine plasma polymer (ALAPP) layer deposited on highly doped p-type silicon wafers either using a pipettor or a
microarray contact printer. Surface-dependant human embryonic kidney (HEK 293 line) cells were cultured onto the
DNA vector loaded ALAPP spots and the plasmid transfection events were detected by fluorescence microscopy. Cell
viability assays, including fluorescein diacetate (FDA) / Hoechst DNA labelling, were carried out to determine the
number of live adherent cells before and after application of a voltage. A protocol was developed to screen for voltage
biases and exposure times in order to optimise transfection efficiency and cell viability. Cross-contamination between the
microarray spots carrying different DNA vectors was also investigated. By application of a voltage of 286 V/cm for 10
ms, transfection efficiency was doubled compared to using only transfection reagent, whilst maintaining a cell viability
of 60-70% of the positive control.
Microarrays, high-throughput devices for genomic analysis, can be further improved by developing materials that are
able to manipulate the interfacial behaviour of biomolecules. This is achieved both spatially and temporally by smart
materials possessing both switchable and patterned surface properties. A system had been developed to spatially
manipulate both DNA and cell growth based upon the surface modification of highly doped silicon by plasma
polymerisation and polyethylene grafting followed by masked laser ablation for formation of a pattered surface with both
bioactive and non-fouling regions. This platform has been successfully applied to transfected cell microarray applications
with the parallel expression of genes by utilising its ability to direct and limit both DNA and cell attachment to specific
sites. One of the greatest advantages of this system is its application to reverse transfection, whereupon by utilising the
switchable adsorption and desorption of DNA using a voltage bias, the efficiency of cell transfection can be enhanced.
However, it was shown that application of a voltage also reduces the viability of neuroblastoma cells grown on a plasma
polymer surface, but not human embryonic kidney cells. This suggests that the application of a voltage may not only
result in the desorption of bound DNA but may also affect attached cells. The characterisation of a DNA microarray by
contact printing has also been investigated.
Tissue engineering and stem cell technologies have led to a rapidly increasing interest in the control of the behavior of mammalian cells growing on tissue culture substrates. Multifunctional polymer coatings can assist research in this area in many ways, for example, by providing low non-specific protein adsorption properties and reactive functional groups at the surface. The latter can be used for immobilization of specific biological factors that influence cell behavior. In this study, glass slides were coated with copolymers of glycidyl methacrylate (GMA) and poly(ethylene glycol) methacrylate (PEGMA). The coatings were prepared by three different methods based on dip and spin coating as well as polymer grafting procedures. Coatings were characterized by X-ray photoelectron spectroscopy, surface sensitive infrared spectroscopy, ellipsometry and contact angle measurements. A fluorescently labelled protein was deposited onto reactive coatings using a contact microarrayer. Printing of a model protein (fluorescein labeled bovine serum albumin) was performed at different protein concentrations, pH, temperature, humidity and using different micropins. The arraying of proteins was studied with a microarray scanner. Arrays printed at a protein concentration above 50 μg/mL prepared in pH 5 phosphate buffer at 10°C and 65% relative humidity gave the most favourable results in terms of the homogeneity of the printed spots and the fluorescence intensity.
The evaluation of cell-material surface interactions is important for the design of novel biomaterials which are used in a
variety of biomedical applications. While traditional in vitro test methods have routinely used samples of relatively
large size, microarrays representing different biomaterials offer many advantages, including high throughput and
reduced sample handling. Here, we describe the simultaneous cell-based testing of matrices of polymeric biomaterials,
arrayed on glass slides with a low cell-attachment background coating. Arrays were constructed using a microarray robot at 6 fold redundancy with solid pins having a diameter of 375 μm. Printed solutions contained at least one
monomer, an initiator and a bifunctional crosslinker. After subsequent UV polymerisation, the arrays were washed and
characterised by X-ray photoelectron spectroscopy. Cell culture experiments were carried out over 24 hours using HeLa
cells. After labelling with CellTracker® Green for the final hour of incubation and subsequent fixation, the arrays were
scanned. In addition, individual spots were also viewed by fluorescence microscopy. The evaluation of cell-surface
interactions in high-throughput assays as demonstrated here is a key enabling technology for the effective development
of future biomaterials.
Manipulating biomolecules at solid/liquid interfaces is important for the development of various biodevices including
microarrays. Smart materials that enable both spatial and temporal control of biomolecules by combining switchability
with patterned surface chemistry offer unprecedented levels of control of biomolecule manipulation. Such a system has
been developed for the microscale spatial control over both DNA and cell growth on highly doped p-type silicon.
Surface modification, involving plasma polymerisation of allylamine and poly(ethlylene glycol) grafting with subsequent
laser ablation, led to the production of a patterned surface with dual biomolecule adsorption and desorption properties.
On patterned surfaces, preferential electro-stimulated adsorption of DNA to the allylamine plasma polymer surface and
subsequent desorption by the application of a negative bias was observed. The ability of this surface to control both
DNA and cell attachment in four dimensions has been demonstrated, exemplifying its capacity to be used for complex
biological studies such as gene function analysis. This system has been successfully applied to living microarray
applications and is an exciting platform for any system incorporating biomolecules.
Control over biomolecule interactions at interfaces is becoming an increasingly important goal for a range of scientific
fields and is being intensively studied in areas of biotechnological, biomedical and materials science. Improvement in the
control over materials and biomolecules is particularly important to applications such as arrays, biosensors, tissue
engineering, drug delivery and 'lab on a chip' devices. Further development of these devices is expected to be achieved
with thin coatings of stimuli responsive materials that can have their chemical properties 'switched' or tuned to stimulate
a certain biological response such as adsorption/desorption of proteins. Switchable coatings show great potential for the
realisation of spatial and temporal immobilisation of cells and biomolecules such as DNA and proteins.
This study focuses on protein adsorption onto coatings of the thermosensitive polymer poly(N-isopropylacrylamide)
(pNIPAM) which can exhibit low and high protein adsorption properties based on its temperature dependent
conformation. At temperatures above its lower critical solution temperature (LCST) pNIPAM polymer chains are
collapsed and protein adsorbing whilst below the LCST they are hydrated and protein repellent.
Coatings of pNIPAM on silicon wafers were prepared by free radical polymerisation in the presence of surface bound
polymerisable groups. Surface analysis and protein adsorption was carried out using X-ray photoelectron spectroscopy,
time of flight secondary ion mass spectrometry and contact angle measurements.
This study is expected to aid the development of stimuli-responsive coatings for biochips and biodevices.
Inorganic/organic hybrid or composite materials have in the past shown novel and interesting properties, which are not observed for the individual components. In this context, the preparation of inorganic/polymeric composites from biodegradable and biocompatible constituents is a new concept, which may be of interest particularly for tissue engineering and drug delivery applications. We describe here the synthesis of nanostructured porous silicon (pSi) and poly(L-lactide) (PLLA) composites. The composites were produced using tin(II) 2-ethylhexanoate catalysed surface initiated ring opening polymerisation of L-lactide onto silanised porous silicon films and microparticles. The subsequent chemical, physiochemical and morphological characterisation was performed using Diffuse Reflectance Infrared Spectroscopy (DRIFTS), X-ray Photoelectron Spectroscopy (XPS), Atomic Force Microscopy (AFM), Differential Scanning Calorimetery (DSC), Thermogravimetric Analysis (TGA) and Contact Angle measurements. DRIFT spectra of the composites showed the presence of bands corresponding to ester carbonyl stretching vibrations as well as hydrocarbon stretching vibrations. XPS analysis confirmed that a layer of PLLA had been grafted onto pSi judging by the low Si content (ca. 3%) and O/C ratio close to that found for PLLA homopolymers. Comparison of the sessile drop contact angle produced by silanised pSi and PLLA grafted onto pSi showed an increase of ca. 40°. This is comparable to the increase in contact angle seen between blank silicon and spin-coated PLLA of ca. 44°. The AFM surface roughness after surface initiated polymerisation increased significantly and AFM images showed the formation of PLLA nanobrushes.
The control over protein adsorption is of major importance for a variety of biomedical applications from diagnostic assays to tissue engineered medical devices. Most research has focused on the prevention of non-specific protein adsorption on solid substrates. Examples for surface modifications that significantly reduce protein adsorption include the grafting of polyacrylamide, poly (ethylene oxide) and polysaccharides. Here, we describe a method for creating surfaces that prevent non-specific protein adsorption, which in addition can be transformed into surfaces showing high protein adsorption on demand. Doped silicon wafers were used as substrate materials. Coatings were constructed by deposition of allylamine plasma polymer. The subsequent grafting of poly (ethylene oxide) aldehyde resulted in a surface with low protein fouling character. When the conductive silicon wafer was used as an electrode, the resulting field induced the adsorption of selected proteins.
Surface modifications were analysed by X-ray photoelectron spectroscopy and atomic force microscopy. The controlled adsorption of proteins was investigated using a colorimetric assay to test enzymatic activity. The method described here represents an effective tool for the control over protein adsorption and is expected to find use in a variety of biomedical applications particularly in the area of biochips.
The recent development of living microarrays as novel tools for the analysis of gene expression in an in-situ environment promises to unravel gene function within living organisms. In order to significantly enhance microarray performance, we are working towards electro-responsive DNA transfection chips. This study focuses on the control of DNA adsorption and desorption by appropriate surface modification of highly doped p++ silicon. Silicon was modified by plasma polymerisation of allylamine (ALAPP), a non-toxic surface that sustains cell growth. Subsequent high surface density grafting of poly(ethylene oxide) formed a layer resistant to biomolecule adsorption and cell attachment. Spatially controlled excimer laser ablation of the surface produced micron resolution patterns of re-exposed plasma polymer whilst the rest of the surface remained non-fouling. We observed electro-stimulated preferential adsorption of DNA to the ALAPP surface and subsequent desorption by the application of a negative bias. Cell culture experiments with HEK 293 cells demonstrated efficient and controlled transfection of cells using the expression of green fluorescent protein as a reporter. Thus, these chemically patterned surfaces are promising platforms for use as living microarrays.
In this work protein patterning has been achieved within a polycarbonate microfluidic device. Channel structures were first coated with plasma polymerized allylamine (ALAPP) followed by the "cloud point" deposition of polyethylene oxide (PEO), a protein repellent molecule. Excimer laser micromachining was used to pattern the PEO to control protein localization. Subsequent removal of a sacrificial layer of polycarbonate resulted in the patterned polymer coating only in the channels of a simple fluidic device. Following a final diffusion bonding fabrication step the devices were filled with a buffer containing Streptavidin conjugated with fluorescein, and visualized under a confocal fluorescent microscope. This confirmed that protein adhesion occurred only in laser patterned areas. The ability to control protein adhesion in microfludic channels leads to the possibility of generating arrays of proteins or cells within polymer microfludics for cheap automated biosensors and synthesis systems.
Two-dimensional control over the surface chemistry of substrate materials is of interest to a wide range of applications from microelectronics to biomedical diagnostics. Here, we describe a general principle for creating spatially controlled surface chemistries by subsequent deposition of thin plasma polymer coatings followed by laser ablation. The creation of surfaces with spatially controlled wettability was used as an example. Plasma polymerization of n-heptylamine produced a hydrophilic surface on silicon wafer substrates while subsequent plasma polymerization of perfluoro-1,3-dimethylcyclohexane produced a hydrophobic surface. Excimer laser ablation at an energy density of 125 mJ/cm2 was used to remove a defined thickness of the two-layer coating, completely removing the upper layer without exposing the substrate material. Excimer laser ablation resulted in two-dimensional control over the surface chemistry with a resolution of ca. 1 μm. Surface modifications were characterized by X-ray photoelectron spectroscopy (XPS) and atomic force microscopy (AFM). Contact angle measurements were used to estimate the wettability of modified surfaces. The method was shown to be suitable for the precise control over the location of droplets containing aqueous solutions. In conclusion, the method described here represents an effective tool for the production of substrates with spatially controlled surface chemistry and wettability.
This paper investigates the ability to modify the surface of silicon wafers for selective cell adhesion and the efficacy of solid phase transfections on the modified surface. Silicon surfaces are first modified by plasma polymerization of allylamine (ALAPP) and subsequent grafting of a protein-resistant layer of poly(ethylene oxide) (PEO) on the plasma polymer surface. Spatially controlled excimer ablation was then used to pattern the graft-copolymer surface for selective cell adhesion. X-ray photoelectron spectroscopy and contact angle measurements confirmed the creation of 2D patterns with different surface chemistry. Cell culture experiments with HEK 293 cells showed that cell attachment is limited to the ablated areas. Furthermore, cells could be transformed with plasmid DNA containing the gene for green fluorescent protein. Therefore, the biochip platform described in this paper, has the potential to be developed into a high-density array for analyzing gene products produced from a matrix of living cells.
Two-dimensional control over the location of proteins on surfaces is desired for a number of applications including diagnostic tests and tissue engineered medical devices. Many of these applications require patterns of specific proteins that allow subsequent two-dimensionally controlled cell attachment. The ideal technique would allow the deposition of specific protein patterns in areas where cell attachment is required, with complete prevention of unspecific protein adsorption in areas where cells are not supposed to attach. In our study, collagen I was used as an example for an extracellular matrix protein known to support the attachment of bovine corneal epithelial cells. An allylamine plasma polymer was deposited on a silicon wafer substrate, followed by grafting of poly(ethylene oxide). Two-dimensional control over the surface chemistry was achieved using a 248 nm excimer laser. Results obtained by XPS and AFM show that the combination of extremely low-fouling surfaces with excimer laser ablation can be used effectively for the production of spatially controlled protein patterns with a resolution of less than 1 micrometers . Furthermore, it was shown that bovine corneal epithelial cell attachment followed exactly the created protein patterns. The presented method is an effective tool for a number of in vitro and in vivo applications.
For many applications, it is essential to be able to control the interface between devices and the biological environment by nanoscale control of the composition of the surface chemistry and the surface topography. Application of molecular thickness coatings of biologically active macromolecules provides predictable interfacial control over interactions with biological media. The covalent surface immobilization of polysaccharides, proteins, and synthetic oligopeptides can be achieved via ultrathin interfacial bonding layers deposited by gas plasma methods, and the multistep coating schemes are verified by XPS analyses. Interactions between biomolecular coatings and biological fluids are studied by MALDI mass spectrometry and ELISA assays. Using a colloid-modified AFM tip, quantitative measurement of interfacial forces is achieved. Comparison with theoretical predictions allows elucidation of the key interfacial forces that operate between surfaces and approaching macromolecules. In this way, it is possible to unravel the fundamental information required for the guided design and optimization of biologically active nanoscale coatings that confer predictable properties to synthetic carriers. We have established for instance the key properties that make specific polysaccharide coatings resistant to the adsorption of proteins, which is applicable to biomaterials, biosensors, and biochips research.
The two-dimensional control of cell adhesion is desired for a number of cell- and tissue culture applications. Thus, a suitable method for the two-dimensional control over surface chemistry, which leads to the display of cell-adhesive and non-adhesive signals is required. In our study, allylamine plasma polymer (ALAPP) deposition has been used to provide a cell-adhesive substrate, while additional grafting of poly(ethylene oxide) (PEO) on ALAPP surfaces has been used to prevent cell adhesion. Two-dimensional control over the surface chemistry was achieved using excimer laser ablation. Ablation experiments were carried out using a 248 nm excimer laser with energy densities of 17 - 1181 mJ/cm2 and 1 - 16 pulses per area. Results obtained by X-ray photoelectron spectroscopy (XPS) and atomic force microscopy (AFM) show controlled thickness ablation of the plasma polymer and the additional PEO graft polymer. Cell culture experiments using bovine corneal epithelial cells show that two-dimensional control of cell adhesion can be achieved by using appropriate masks in the laser beam.