Morphology and adhesion of biomolecules on silicon based surfaces
Introduction
Proteins [1] on silicon based surfaces are of extreme importance in various applications including silicon microimplants, various microdevices such as biosensors, and therapeutics [2], [3], [4], [5], [6]. Silicon is a commonly used substrate in microimplants, but it can have undesired interactions with the human immune system. Therefore, to mimic a biological surface, protein coatings are used on silicon based surfaces as a passivation layer, so that these are compatible in the body and minimize an inflammatory response. Whether this surface treatment is applied to a large implant or a microdevice, the function of the protein passivation is obtained from the nanoscale 3D structural conformation of the protein. Proteins are also used in microdevices because of their functional specificity. For biosensor applications, the extensive array of protein activities provides a rich supply of operations that may be performed at the nanoscale. Many antibodies (proteins) have an affinity to specific protein ligands. For example, pathogens (disease causing agents, e.g., virus or bacteria) trigger production of antigens which can be detected when bound to a specific antibody on the biosensor. The specific binding behavior of proteins that has been applied to laboratory assays may also be redesigned for in vivo use as sensing elements of a microdevice. The epitope-specific binding properties of proteins to various antigens are useful in therapeutics. Adhesion between the protein and substrate affect reliability of an application. Among other things, morphology of the substrate affects the adhesion. Furthermore, for in vivo environments, the proteins on the biosensor surface have to have abrasion resistance during the direct contact with the tissue and circulatory blood flow without washing off.
An example of a biosensor under development in our laboratory is based on a field-effect transistor (FET) and is shown in Fig. 1. FETs are sensitive to the electrical field produced in the channel due to the charge at the surface of the gate insulator. In this sensor, the gate metal of a metal-oxide semiconductor field effect transistor (MOSFET) is removed and replaced with a protein whose cognate is the component that is meant to be sensed. The binding of a protein and its cognate produces a change in the effective charge which creates a change in the electrical field. This electrical field change may produce a measurable change in the current flow through the device.
Atomic force microscopy (AFM) has been used to study morphology of surfaces as well as measurement of adhesion [7], [8]. Since biological surfaces are soft, imaging either in the contact mode or in a dry environment is not preferred. Tapping mode AFM is commonly used [9], [10], [11], [12], [13]. For high resolution imaging, immobilization of the biological molecule is necessary to study biologically modified surfaces [14]. In some cases, it is very difficult to distinguish biomolecules from the surface features of a rough surface and in this case phase imaging AFM is helpful to distinguish biomolecules clearly from the surface height distribution [15]. AFM can also be used to study the mobility of biomolecules like DNA [16]. In order to study mobility of biomolecules, they are injected on the surface during imaging so that imaging can be performed before immobilization. Functionalized AFM probes, sometimes referred to as nanobiosensors, are used to detect and image specific molecules which interact with the biomolecules immobilized on AFM tip [10], [17]. These probes are also used to study the bonding between the biological molecules and the substrate surfaces of interest [18], [19], [20]. When surfaces are imaged under physiological conditions, liquid buffers used during the imaging affects the data [21]. (A buffer is a solution with a specified pH that resists change in pH upon addition of small amounts of acid or base. Ionic buffers are required to stabilize biomolecules in their natural structure.)
Although AFMs have been used to study morphology and adhesion, none of the above research has been dedicated to the morphological changes of a substrate surface with treatments that are commonly used during surface modification. So, very little is known about morphological changes as a function of surface treatments. Adhesion properties between the substrate and biomolecules are also not well understood.
The objective of this research is to study the step-by-step morphological changes and the adhesion of a protein model on silicon based surfaces with respect to surface treatment using atomic force microscopy. Some approaches to improve adhesion between biomolecules and silicon based surfaces are discussed. Even though this study was initially motivated by the sensor context, the method of protein binding to surfaces has much broader applications. The information on binding obtained from these studies could be tuned for use with other surfaces and other proteins.
Section snippets
Surface morphology
Imaging was carried out using a commercial AFM (Dimension 3000, Digital Instruments, Santa Barbara, CA) in tapping mode in a liquid environment [8]. A fluid cell was used for measurements in a liquid environment. A schematic of a fluid cell is shown in Fig. 2. The working principle of a fluid cell is similar to that for normal tapping mode except the manner in which tip is excited. In the tapping mode in air, the cantilever tip assembly is vibrated by a piezo mounted above it, and the
Surface morphology and effect of buffers on surface
To understand the morphological changes in the silicon surface with respect to surface treatment, a step by step morphological analysis after every surface treatment was conducted. Fig. 4(a) shows the basic AFM morphology of various surfaces imaged in air and liquid environments. The supplied silicon surface was very smooth, and the RMS roughness increased with oxidation. The surface RMS roughness over an area of 1 μm × 1 μm and peak to valley distance values of all samples are listed in Table 1.
Conclusions
To improve adhesion between silica surface and biomolecules, two kinds of surface modification approaches have been studied, direct adsorption and chemical linkage (e.g., biotin–streptavidin). To prepare a surface for chemical linkage, it is first cleaned, hydroxylated and silanized with various buffers. The surface is then coated with chemical layers to enhance adhesion with the biomolecules of interest. The duration of these surface modification processes lie between 15 min and 2 h. During a
Acknowledgement
The financial support for this research was provided by the industrial membership of the Nanotribology Laboratory for Information Storage and MEMS/NEMS (NLIM). Nanopatterning of silica samples was done in Prof. L.J. Brillson’s laboratory.
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