A description of the formation of a polymer microarray using an on-chip photopolymerization technique. The high throughput surface characterization using atomic force microscopy, water contact angle measurements, X-ray photoelectron spectroscopy and time of flight secondary ion mass spectrometry and a cell attachment assay is also described.
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 format1. 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 criterion3-5. 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 interaction6. 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 materials5,3.
1. Preparation of low-fouling background
2. Preparation of monomer solution
3. Polymer microarray formation
The typical procedure for polymer microarray formation is depicted schematically in Figure 1.
4. High throughput surface characterization (HTSC)
A general scheme of the HTSC techniques is shown in Figure 3. Central to the automated, high throughput approach is the alignment of the polymer microarray with the characterization apparatus. In all cases this is achieved using a camera that gives a top view of the array. Initially, the array is rotated to align with the X-Y movement of the stage. A corner spot of the array is then located and designated specific coordinates. The position of each polymer spot can then be predicted using the dimensions of the array.
5. Bacterial assay
The array can be exposed to many different biological assays including attachment and proliferation of stem cells, other cells types and bacteria 3,10,4. Here we describe a bacterial attachment assay, which is shown schematically in Figure 4.
6. Representative Results
The conditions of printing have been optimized to print the highest quality polymer microarrays. The humidity should be kept at between 30-40%. The delamination of polymer spots in aqueous environments was observed frequently for arrays printed at a humidity below 30%, suggesting that this humidity is insufficient to swell the pHEMA layer and allow for the physical entrapment of the polymer to the substrate. The humidity can be increased further to alter the diameter of the polymer spots, but this is dependent on the monomer chemistry. For example, where equal volumes of polymerization solution were printed and as humidity was increased from 40 to 80 % the spot diameter decreased from 430 μm to 370 μm for a monomer containing a hydrophilic ethylene glycol moiety equal volume whilst for a monomer containing a hydrophobic aliphatic carbon ring structure the spot diameter increased from 290 μm to 350 μm (Figure 5).
The degree of polymerization can be monitored using Raman spectroscopy to measure the C=C Raman shift that is detected at 1640 cm-1, which should be normalized with the C=O Raman shift at 1720 cm-1. The Raman spectra was measured for polymer spots polymerized for varied UV exposure (Figure 6). The C=C:C=O ratio decreased as UV exposure increased from 0 to 50 s, whereupon no further decrease in the C=C:C=O ratio was observed with further UV irradiation (Figure 6). Raman spectra were also measured for polymer spots polymerized at varied O2 level and the C=C Raman shift was observed as the O2level was decreased to 2000 ppm, however no further reduction was observed for an O2 level below this (Figure 7A). Raman spectroscopy also demonstrated the ability of the vacuum extraction step to remove unpolymerized monomer. Prior to vacuum extraction the C=C Raman shift was greater for the polymer polymerized at 3300 ppm compared with 2000 ppm (Figure 7A), however, after vacuum extraction the height of the Raman shift is indistinguishable (Figure 7B), suggesting all unpolymerized monomer has been removed during the vacuum extraction step. To summarize, polymerization conditions include a humidity of 30-40%, UV exposure greater than 50 s at an O2 level below 2000 ppm with a vacuum extraction step after printing for 7 days.
After printing and vacuum extraction the success of the polymerization of polymer spots can be assessed by simple light microscopy to identify and anomalous spot morphologies. Typically, spots should appear circular and uniform, as shown in Figure 8 on the left. The likely cause for a change in geometry is a damaged or unclean pin. For a small number of monomer combinations we have observed misshapen spots, for example a central spot with a satellite of small spots, shown in Figure 8 on the right, or a fried egg shape where there is a central spot on top of large, flatter spot. This may be caused by phase separation prior to printing relating to differences in the viscosity, hydrophilicity, volatility or surface tension of the monomers and suggests that the monomer combination is not compatible with this format. Additional chemical mapping of polymer spots by techniques such as ToF-SIMS is also an important and sometimes necessary quality control step to determine the distribution of the materials’ chemistries across the spots and the array. This technique can identify excessive spreading of some materials not visible by light microscopy and identify phase separation within individual polymer spots.
Figure 1. Schematic depicting the various steps involved in the formation of a polymer spot.
Figure 2. Schematic of the methodology of pin printing involving initially loading the pin with monomer in a source plate and then depositing the monomer onto a substrate by making contact. The pin is controlled by an X-Y-Z robotic arm. The inset shows a typical image of the autofluorescence from an array after production.
Figure 3. Schematic highlighting the techniques associated with HTSC and also bioassays applied to the study of polymer microarrays.
Figure 4. Schematic of the bacterial attachment assay.
Figure 5. Polymer spot diameter printed at varied humidity for two different monomers: 4-tert-butylcyclohexyl acrylate and di(ethylene glycol) ethyl ether methacrylate.
Figure 6. The ratio of the Raman intensity for the C=C Raman shift at 1640 cm-1 and the C=O Raman shift at 1720 cm-1 from polymer spots of 4-tert-butylcyclohexyl acrylate with varied UV exposure. The error bars equal one standard deviation (n = 3).
Figure 7. The Raman spectra measured for polymer spots of 4-tert-butylcyclohexyl acrylate printed at varied O2levels, indicated to the left of each spectrum, (A) before and (B) after vacuum extraction. The ratio of the Raman intensity for the C=C Raman shift at 1640 cm-1 and the C=O Raman shift at 1720 cm-1 is shown to the right of each spectrum.
Figure 8. A light microscopy image of two polymer spots. The spot on the left shows a well formed spot, whilst the spot on the right is an example of a spot containing a very unevenly distribution of monomer. The scale bar is 500 μm.
Polymer microarrays have been successfully used for the discovery of new materials by screening hundreds of novel polymer in a biological assay and identifying ‘hit’ materials that can subsequently be scaled up to useful devices. In this case, the surface characterization described may be employed subsequent to the biological assay and exclusively on the ‘hit’ materials to study those materials in more detail. This strategy may be of interest if HTSC is not available to the experimentalist employing this approach. However, to fully utilize polymer microarrays to study biological-material interactions the entire array of hundreds of materials should be analyzed prior to biological assays using HTSC methodologies, which can subsequently be used to observe general structure-function trends.
Contact printing relies on the metal pin sliding up and down freely within the pin holder. Pin and pin holder cleanliness is paramount is ensuring printing occurs successfully and should be rigorously undertaken. Before commencing a printing run the appropriate movement of the pin within the pin holder can be tested by performing a dry run, with no monomers present. The cleaning step should continue until the pin movement is achieved reproducibly.
Considerable thought should go into the design of the monomer mixture. In order to easily produce a combinatorial library of polymers, hundreds of copolymers are formed by mixing a few monomers at different ratios. Typically we produce 576 member libraries as this forms a 24 x 24 array, which is suitable for the geometry of a glass slide. In order to produce a combinatorial library that explores the most combinatorial space the easiest method is to mix 24 monomers pairwise at a 2:1 ratio. Alternatively, the inclusion of compositional gradients within the array are useful for enabling the observations of trends, which allows optimal monomer compositions to be determined. As an example of this 22 monomers can be used as the first component in a co-monomer mixture that is sequentially diluted with 1 of 6 second components. If 5 dilutions are used, for example mixing the first and second components at ratios of 90:10, 75:25, 50:50, 25:75 and 10:90, this would result in 488 unique copolymer solutions. To bring the total up to 576, replicates of the homopolymers of the monomers used can be introduced, which often is an important reference sample. 576 monomer solutions should be dispensed into 2 384 well plates. For programming the robot it is easier to have two identical plates in terms of the position of the monomers, thus, the monomer solutions should be split evenly between the two plates.
A significant amount of time can be saved in the preparation of the source plates by the use of multichannel pipettes, and the design of source plates should be determined in order to exploit the use of the multichannel pipettes.
To achieve automated HTSC of the arrays the spot position must be successfully aligned with the characterization apparatus. Typically the pitch of an acrylate array is 500-1000 μm and the polymer spot diameter is 300 μm. Most X-Y stages have a resolution below 10 μm, thus there is adequate tolerance for the surface characterization apparatus to reliably access the array positions once the correct dimensions have been input to the sample positioning software. The limitation to the automated positioning is in fact the accurate printing of the array. To ensure accurate printing it is important to prevent movement of the substrate on the printing stage either using vacuum suction or spring clamps together with appropriate slide dimensions (note that both a US and EU standard slide size exist).
ToF-SIMS is an extremely surface sensitive technique that will observe any contamination on samples. Thus, upmost care must be taken to avoid contact with the surface. Samples should only be handled, but the surface of interest not contacted with, with clean gloves (preferably polyethylene) and with freshly cleaned tweezers. We typically wash with chloroform and hexane. Sample storage prior to measurements is best done in a sample holder that holds the slides apart, for example the 5 slide holder or 20 slide holder.
The arrays are designed specifically to be compatible with many biological assay formats and readouts, that is, the substrate used is a microscope slide ideally suited to fluorescence scanners and light microscopes. This means the format is well suited to exploring many material-biological interactions. Furthermore, the format allows hundreds of materials to be screened in parallel. This allows many more materials to be screened than conventional methods whereby each new material chemistry is screened individually. The increased scope for biological-material interactions allows for the elucidation of mechanisms of biological surface interactions, as well as finding the optimal material for a given application.
The authors have nothing to disclose.
Funding from the Wellcome Trust is kindly acknowledged (grant number 085245/Z/08/Z). Nottingham Nanotechnology and Nanoscience Centre is kindly acknowledged for giving access to the Raman system and for the East Midlands Development Agency for funding this equipment.
Name of the reagent/equipment | Company | Catalogue/Model number |
Epoxy slides | Genetix | K2652 |
Contact printer | Biodot | XYZ3060 Platform |
Metal pin | ArrayIt | 946MP6B |
ToF-SIMS instrument | ION-TOF | |
XPS instrument | Kratos | |
WCA apparatus | Krüss | DSA 100 |
AFM | Bruker | Dimension Icon |
RPMI-1640 cell culture media | Sigma-Aldrich | R0883 |
SYTO17 | Invitrogen | S-7579 |