Substrates of protein microarray

1.2.2.1.The choice for solid substrates of protein microarray

One of the most important factors that determine the performance of protein microarrays is the solid substrate on which the proteins are immobilized. The selected materials used as the solid support of protein microarray should not only efficiently immobilize proteins on its surface but also keep their best biological activities. Generally, the solid support for the protein microarray should meet the requirements as following:

Firstly, the solid support should provide optimal binding conditions with high binding capacities of proteins and keep the stability of the resulting biosensor layer. Because protein has no specific uniform adhesion due to its complex structure, the immobilization on the support depends on the properties of each protein.

Secondly, materials must be adapted to the detection methods used. For instance, a label free detection as SPR (Surface Plasmon Resonnance) requires a dielectric/metal interface with two different reflexion indices. In this case, a thin metal layer (gold or silver) must be deposited on the surface of glass slide. For electrochemical detection, solid support must be conducting (metal or semiconductor materials as Si/SiO2 are required for potentiometric measurement). Besides, the physical properties of supports should have no great effects on the detection information of biosensor layer and low intrinsic signals. For example, auto- fluorescence or background of the support is avoided if using fluorescence scanning as the detection system.

At last, the solid support must be suitable for high-throughput manufacturing and screening procedures. This includes rapid and inexpensive production in high quantities, ease of handling during storage and preparation procedures as well as high reproducibility. The material should also be compatible with all fabrication steps including harsh washing conditions or micro-technological steps. Thus, it is required to provide a non-denaturing environment to avoid the loss of activity and binding sites during the process of immobilization, because the objective of protein microarray technology is the investigation of interactions between proteins with their biological activities.

From the viewpoint of physical structure, the solid substrate includes 2-dimension (2D, flat) or 3-dimension (3D, with microstructure such as microwell on the surface). The flat 2- dimension (2D) slides are commonly used as the solid support for manufacturing protein microarray due to the low cost and easy treatment. However, protein microarray based on 2D supports normally can just be put in contact with only one solution. In order to perform multiplex analysis of a set of sample analyte, Mazurczyk et al [91] developed one process to elaborate 3-dimension (3D). By photolithography onto glass slides, they groove microwells with depths exceeding 100 µm and surface roughness below 10 nm (Figure 1-6). These microstructured slides not only retain surface properties of the original glass substrate, but also do not increase the fluorescence background level. This makes it possible to use the existing methods of glass surface functionalisation for protein immobilization and the classical fluorescence scanner for detection.

1.2.2.2.Typical substrates

Previously, filterable membranes, such as polyvinylidene fluoride (PVDF) and cellulose nitrate membranes were chosen as the support for high-density protein microarrays, due to their low-price, easy-preparation, and direct immobilization of large amount of protein without surface modification [92]. The interaction between the surface and proteins is physical adsorption, which represents the simplest process of protein binding, although it is rather uncontrollable. Close proximity between the adsorptive surface and the reactive site of protein could have unfavourable effects on the biological activity towards its ligands. Besides, the surface of the solid may also be susceptible to exchanging adsorbed protein due to the surrounding solution and non-specific adsorption could be also a problem. Therefore, they have been gradually replaced by other solid supports due to their too many uncontrolled parameters. However, some groups now arrayed proteins on polymer support primarily for optical and economical reasons [93, 94]. Cyclic polyolefin slides, like Zeonor representing a class of new polymeric materials with excellent optical and mechanical properties, were employed to immobilize antibody [93]. Glycidyl methacrylate (GMA)-modified polyethylene terephthalate (PET) plastic, which can introduce high density of epoxy groups to PET surface by grafting GMA photopolymer, was described as good solid support to manufacture high performance protein microarrays [94].

Glass is a popular material as solid support for protein microarray, primarily due to its low fluorescence, transparency, low cost, and resistance to high temperature [95]. Glass surfaces can be modified by silane chemistry introducing specific functional group such as amino groups, epoxide, carboxylic acid and aldehyde, which can directly react with protein

by physisorption/chemisorption or further covalent bind with other biocompatible chemistries (e.g. chitosan) to generate novel surfaces. In addition, glass offers a number of practical advantages over porous membranes and gel pads, which is easy to handle and adaptable to automatic readers. Currently, two major categories of microarray slides exist: gel-coated surfaces, such as polyacrylamide [96] or agarose [97] and non-gel-coated modified glass surfaces, such as aldehyde [32], poly-L-lysine [98], or nickel-coated slides [99]. In addition, gold film deposited on the solid support is commonly employed as the protein microarray substrate with the SPR detection [100].