Introduction 1 2 3 4 6 7 6 12 1 3 5 8 10 −4 In this report, we review applications of SEIRA in which biochemical processes were studied. Because SEIRAS was introduced to the field of biomolecules only recently, we consider it worthwhile to start with practical aspects of the method. Experimental considerations Preparation of thin metal-film substrates 11 −1 2 11 12 13 14 17 w v 4 −1 4 −1 2 3 −1 2 2 3 −1 4 w v is put on the Si surface for 60–90 s. −1 2 4 16 17 18 19 Removal of the metal film from the supporting substrate 2 2 2 Geometry and optical configuration 1 1 2 2 1 11 20 Fig. 1 a b arrows i ii iii c  −1 −1 1 21 22 22 Chemical modification of the metal film surface 23 a thiol group that is spontaneously adsorbed by the metal electrode; a spacer group with different lengths of alkyl chain; and a functional headgroup pointing toward the bulk solution. The CME surface can be conveniently prepared by the so-called “SAM (self-assembled monolayer) method” in which the SEIRA-active metal substrate is immersed in a solution containing the crosslinker molecule (normally for between 10 min and 2 h, depending on size of the molecule). The SAM of the crosslinker is formed spontaneously by quasi-covalent bonding between the sulfur and the metal surface. The substrate is then thoroughly rinsed with the solvent so that a SAM of the crosslinker remains on the metal substrate. 2+ 24 25 Applications Studies of nucleic acids and DNA 26 27 28 29 −1 30 31 31 Immunoassays based on SEIRAS S N 32 −1 −1 19 19 18 SEIRAS to probe protein functionality The metal used for surface enhancement, can also be used as an electrode. The enzymatic reactions of many biological systems, especially those of membrane proteins, are driven by an electrochemical gradient across the cell membrane. Such a system can be artificially reproduced on an electrode surface to mimic the physiological properties of a biological membrane. 33 2 14 14 Fig. 2 a b c d 14  Studies of molecular and protein recognition The acute sensitivity of SEIRAS is particularly useful when the technique is applied to studies of membrane proteins. Handling of the membrane proteins usually requires great care, because of their sensitivity to degeneration as soon as they are separated from the native lipid bilayer. The large size of the proteins also makes it difficult to control their orientation. A successful strategy for immobilization of membrane proteins is to attach the purified protein, by using the selective affinity of a genetically introduced histidine tag (His-tag), to a nickel-chelating nitrilotriacetic acid (Ni-NTA) monolayer (SAM) self-assembled on a chemically modified gold surface. The lipid environment of the surface-anchored membrane protein is subsequently restored by in-situ dialysis of the detergent around the amphiphilic membrane protein. This approach results in orientated immobilization and reconstitution of the native matrix, which enhances the stability of the membrane protein and restores full functionality. 24 25 Rhodobacter sphaeroides 34 3 35 36 24 25 3 14 R. sphaeroides 37 Fig. 3 a b c a b d  38 This methodology is a general approach for immobilization of proteins, because the introduction of affinity tags is routine with modern genetic techniques. Other tags beyond the His-tag may also be an option. Thus, orientational control of protein adsorption on a solid surface can be conveniently achieved. An oriented sample is mandatory when the vectorial function of membrane proteins is addressed. Many membrane proteins are asymmetric in their functionality, because they translocate ions or solutes preferentially in one direction or because their stimulant, e.g. ligand, binding partner protein, membrane potential, etc., affects the protein from one side only. Summary and outlook Despite the accomplishments reported in this article, studies of biomaterials by SEIRA is still in its infancy. Potential-induced difference spectroscopy using SEIRA is a promising possibility for study of the functionality of proteins. The stimulus in such studies is not limited to the electric trigger, as reviewed here, but can also be light illumination, temperature jump, or chemical induction. Many protein functions can be addressed by these means and the mechanism of action of these molecular machines may be resolved down to the level of a single bond. 16