The discovery of novel biomaterials that are optimized for a specific biological application is readily achieved using polymer microarrays, which allows a combinatorial library of materials to be screened in a parallel, high throughput format ¹. Herein is described the formation and characterization of a polymer microarray using an on-chip photopolymerization technique ². This involves mixing monomers at varied ratios to produce a library of monomer solutions, transferring the solution to a glass slide format using a robotic printing device and curing with UV irradiation. This format is readily amenable to many biological assays, including stem cell attachment and proliferation, cell sorting and low bacterial adhesion, allowing the ready identification of 'hit' materials that fulfill a specific biological criterion ^3-5. Furthermore, the use of high throughput surface characterization (HTSC) allows the biological performance to be correlated with physio-chemical properties, hence elucidating the biological-material interaction ⁶. HTSC makes use of water contact angle (WCA) measurements, atomic force microscopy (AFM), X-ray photoelectron spectroscopy (XPS) and time-of-flight secondary ion mass spectrometry (ToF-SIMS). In particular, ToF-SIMS provides a chemically rich analysis of the sample that can be used to correlate the cell response with a molecular moiety. In some cases, the biological performance can be predicted from the ToF-SIMS spectra, demonstrating the chemical dependence of a biological-material interaction, and informing the development of hit materials ^5,3.

Biosensors & Bioelectronics

The manipulation of biomolecules at solid/liquid interfaces is important for the enhanced performance of a number of biomedical devices, including biochips. This study focuses on the spatial control of surface interactions of DNA as well as the electro-stimulated adsorption and desorption of DNA by appropriate surface modification of highly doped p-type silicon. Surface modification by plasma polymerisation of allylamine resulted in a surface that supported DNA adsorption and sustained cell attachment. Subsequent high-density grafting of poly(ethylene oxide) formed a low fouling layer resistant to biomolecule adsorption and cell attachment. Spatially controlled excimer laser ablation of the surface produced patterns of re-exposed plasma polymer with high-resolution. 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. Furthermore, the concept presented here was investigated for use in transfection chips. Cell culture experiments with human embryonic kidney cells, using the expression of green fluorescent protein as a reporter, demonstrated efficient and controlled transfection of cells. Electro-stimulated desorption of DNA was shown to yield significantly enhanced solid phase transfection efficiencies to values of up to 30%. The ability to spatially control DNA adsorption combined with the ability to control the binding and release of DNA by application of a controlled voltage enables an advanced level of control over DNA bioactivity on solid substrates and lends itself to biochip applications.

Trends in Biotechnology

Many biological events, such as cellular communication, antigen recognition, tissue repair and DNA linear transfer, are intimately associated with biomolecule interactions at the solid-liquid interface. To facilitate the study and use of these biological events for biodevice and biomaterial applications, a sound understanding of how biomolecules behave at interfaces and a concomitant ability to manipulate biomolecules spatially and temporally at surfaces is required. This is particularly true for cell microarray applications, where a range of biological processes must be duly controlled to maximize the efficiency and throughput of these devices. Of particular interest are transfected-cell microarrays (TCMs), which significantly widen the scope of microarray genomic analysis by enabling the high-throughput analysis of gene function within living cells. This article reviews this current research focus, discussing fundamental and applied research into the spatial and temporal surface manipulation of DNA, proteins and other biomolecules and the implications of this work for TCMs.

Biomacromolecules

The fabrication and characterization of chemical patterns using a technique that can be readily integrated with methods currently used for the formation of microarrays is presented. A high density poly(ethylene glycol) coating was deposited on glass slides as a background exhibiting low cell attachment properties. Phenylazide modified polymers were then printed on this background. UV irradiation of these polymer arrays resulted in the cross-linking of the polymer spots and their covalent attachment to the surface. Cell attachment was shown to follow the resultant surface chemistry pattern. Furthermore, the use of a robotic contact printer enabled the facile deposition of DNA microarrays on top of and aligned with the polymer microarrays. A transfected cell microarray was generated in this way, demonstrating not only the ability of this platform to limit cell attachment to specific regions, but the suitability for chip-based functional genomics, in particular, and high density cell assays in general.

Langmuir : the ACS Journal of Surfaces and Colloids

Polymer microarrays provide a high-throughput format in which to assess biointerfacial interactions. This endeavor greatly assists with the development of advanced biomaterials. In order to increase the scope of this platform technology, the development of analytical tools that are compatible with the microarray format and are capable of analyzing biomolecular interactions in high throughput is needed. Here, we show that surface plasmon resonance imaging (SPRi) is such a tool. SPRi enables spatially resolved, surface sensitive, label free, real-time analysis of multiple surface-biomolecular interactions in parallel. In order to demonstrate this, we first printed phenylazide-modified polymers onto a slide coated with a low fouling base polymer. UV irradiation of the slide resulted in the cross-linking of the printed polymer spots to the surface. SPRi was then employed to study the adsorption and desorption of bovine serum albumin, collagen, and fibronectin to these adhesive microarray spots. The spots were also incubated with an adherent cell line, enabling insight into the underlying mechanisms of cell attachment to the polymers studied. For the system analyzed here, electrostatic interactions were shown to dominate cell attachment.

Biomaterials

Many fundamental biological processes, including early embryo development, immune responses and the progression of pathogens, are mediated by gradients of biological molecules. Understanding these vital physiological processes requires the development of biomaterial platforms that can mimic them in-vitro. Such platforms include laboratory generated surface gradients of biological molecules. In this work, we report a method for the generation of surface gradients of two proteins. We used a surface grafting density gradient of polyethylene glycol (PEG) to control protein adsorption. In addition, we used protein size as a tool to control the position and the adsorbed amount of both proteins. To demonstrate our concept, we used fibrinogen as an example of a large protein and lysozyme as an example of a small protein. However, we speculate that the same strategy could be extended to any other pair of large and small proteins. We used X-ray photoelectron spectroscopy and sessile drop contact angle measurements to determine the chemical composition and wettability of the gradients. Protein adsorption was studied by surface plasmon resonance imaging.

Biomaterials

The high throughput discovery of new bio materials can be achieved by rapidly screening many different materials synthesised by a combinatorial approach to identify the optimal composition that fulfils a particular biomedical application. Here we review the literature in this area and conclude that for polymers this process is best achieved in a microarray format, which enable thousands of cell-material interactions to be monitored on a single chip. Polymer microarrays can be formed by printing pre-synthesised polymers or by printing monomers onto the chip where on-slide polymerisation is initiated. The surface properties of the material can be analysed and correlated to the biological performance using high throughput surface analysis, including time-of-flight secondary ion mass spectrometry (ToF-SIMS), X-ray photoelectron spectroscopy (XPS) and water contact angle (WCA) measurements. This approach enables the surface properties responsible for the success of a material to be understood, which in turn provides the foundations of future material design. The high throughput discovery of materials using polymer microarrays has been explored for many cell-based applications including the isolation of specific cells from heterogeneous populations, the attachment and differentiation of stem cells and the controlled transfection of cells. Further development of polymerisation techniques and high throughput biological assays amenable to the polymer microarray format will broaden the combinatorial space and biological phenomenon that polymer microarrays can explore, and increase their efficacy. This will, in turn, facilitate the discovery of optimised polymeric materials for many biomaterial applications.

Nature Materials

Both human embryonic stem cells and induced pluripotent stem cells can self-renew indefinitely in culture; however, present methods to clonally grow them are inefficient and poorly defined for genetic manipulation and therapeutic purposes. Here we develop the first chemically defined, xeno-free, feeder-free synthetic substrates to support robust self-renewal of fully dissociated human embryonic stem and induced pluripotent stem cells. Material properties including wettability, surface topography, surface chemistry and indentation elastic modulus of all polymeric substrates were quantified using high-throughput methods to develop structure-function relationships between material properties and biological performance. These analyses show that optimal human embryonic stem cell substrates are generated from monomers with high acrylate content, have a moderate wettability and employ integrin alpha(v)beta(3) and alpha(v)beta(5) engagement with adsorbed vitronectin to promote colony formation. The structure-function methodology employed herein provides a general framework for the combinatorial development of synthetic substrates for stem cell culture.

Biofabrication

The spatial control over biomolecule- and cell-surface interactions is of great interest to a broad range of biomedical applications, including sensors, implantable devices and cell microarrays. Microarrays in particular require precise spatial control and the formation of patterns with microscale features. Here, we have developed an approach specifically designed for transfected cell microarray (TCM) applications that allows microscale spatial control over the location of both DNA and cells on highly doped p-type silicon substrates. This was achieved by surface modification, involving plasma polymerization of allylamine, grafting of poly(ethylene glycol) and subsequent excimer laser ablation. DNA could be delivered in a spatially defined manner using ink-jet printing. In addition, electroporation was investigated as an approach to transfect attached cells with adsorbed DNA and good transfection efficiencies of approximately 20% were observed. The ability of the microstructured surfaces to spatially direct both DNA adsorption and cell attachment was demonstrated in a functional TCM, making this system an exciting platform for chip-based functional genomics.

Biomaterials

High throughput materials discovery using combinatorial polymer microarrays to screen for new biomaterials with new and improved function is established as a powerful strategy. Here we combine this screening approach with high throughput surface characterization (HT-SC) to identify surface structure-function relationships. We explore how this combination can help to identify surface chemical moieties that control protein adsorption and subsequent cellular response. The adhesion of human embryoid body (hEB) cells to a large number (496) of different acrylate polymers synthesized in a microarray format is screened using a high throughput procedure. To determine the role of the polymer surface properties on hEB cell adhesion, detailed HT-SC of these acrylate polymers is carried out using time of flight secondary ion mass spectrometry (ToF SIMS), X-ray photoelectron spectroscopy (XPS), pico litre drop sessile water contact angle (WCA) measurement and atomic force microscopy (AFM). A structure-function relationship is identified between the ToF SIMS analysis of the surface chemistry after a fibronectin (Fn) pre-conditioning step and the cell adhesion to each spot using the multivariate analysis technique partial least squares (PLS) regression. Secondary ions indicative of the adsorbed Fn correlate with increased cell adhesion whereas glycol and other functionalities from the polymers are identified that reduce cell adhesion. Furthermore, a strong relationship between the ToF SIMS spectra of bare polymers and the cell adhesion to each spot is identified using PLS regression. This identifies a role for both the surface chemistry of the bare polymer and the pre-adsorbed Fn, as-represented in the ToF SIMS spectra, in controlling cellular adhesion. In contrast, no relationship is found between cell adhesion and wettability, surface roughness, elemental or functional surface composition. The correlation between ToF SIMS data of the surfaces and the cell adhesion demonstrates the ability to identify surface moieties that control protein adsorption and subsequent cell adhesion using ToF SIMS and multivariate analysis.

Journal of Drug Targeting

The application of high throughput surface characterization (HTSC) to the analysis of polymeric biomaterial libraries is an important advancement for the discovery and development of new biomedical materials and is the focus of this review. The potential for HTSC to identify structure/activity relationships for large libraries of materials can be utilized to accelerate materials discovery as well as providing insight into the underlying biological-material interactions. Furthermore, the correlations identified between surface chemical structure and cellular behavior could not have been predicted by a rational design approach based simply on review of bulk structure, which demonstrates the importance of HTSC in the assessment of cell-material and cell-biomolecular interactions that are dependent on surface properties.

Methods in Molecular Biology

DNA, protein and cell microarrays are increasingly used in a multitude of bioassays. All of these arrays require substrates that are suitable for the immobilisation and display of arrayed probe molecules whilst at the same time resisting non-specific interactions of biomolecules with the substrate in areas between printed spots. To meet these conflicting requirements, three different approaches have been developed, all of which were based on low-fouling, high-density poly(ethylene glycol) (PEG) background coatings. In the first approach, the coating was based on allylamine plasma polymerisation (ALAPP) and the subsequent high-density grafting of PEG, followed by the generation of a surface chemical pattern using laser ablation. In the second approach, a photoreactive polymer was printed on the same ALAPP-PEG background. The third approach was based on ALAPP deposition followed by the formation of a multifunctional layer by spin coating a PEG-based polymer that also displayed epoxy groups. The successful demonstration of DNA, protein and cell microarrays has been achieved on each of these coatings.

Journal of Materials Science. Materials in Medicine

Three different formulations comprising two drugs, felodipine and hydrochlorothiazide (HCT) and two polymers, poly(vinyl pyrolidone) (PVP) and poly(lactic-co-glycolic acid) (PLGA) were inkjet printed as micro-dot arrays and analysed on an individual micro-spot basis by time-of-flight secondary ion mass spectrometry (ToF-SIMS). For the HCT/PLGA formulation, the spots showed heterogeneity of the drug and other chemical constituents. To further investigate these heterogeneities, multivariate curve resolution was applied to the ToF-SIMS hyperspectral image datasets. This approach successfully identified distinct chemical components elucidating the HCT, PLGA, substrate material, and contaminants based on sulphur, phosphorous and sodium chloride. Spots printed using either of the drugs with PVP exhibited full substrate coverage and a uniform distribution of the active ingredient along with all other constituents within the printed spot area. This represents the preferred situation in terms of stability and controlling the release of a drug from a polymer matrix.