Approaches to the immobilization of proteins at surfaces for analysis by scanning tunneling microscopy

Abstract

Bovine liver catalase has been immobilized at surfaces by three separate methods. Firstly, catalase molecules were sprayed onto mica and coated with a platinum/carbon film. Images of single molecules showing submolecular structure were obtained, exhibiting a close correlation with data obtained by X-ray crystallography and electron microscopy. Secondly, catalase molecules were thiolated using Traut's reagent (2-iminothiolane) and adsorbed onto gold surfaces. Images of monolayers of the adsorbed protein were obtained, and the effects on the adsorbate of decreasing the tunnel gap resistance were investigated. Thirdly, 3-mercaptopropanoic acid was chemisorbed onto gold surfaces, and coupled to catalase molecules using a water-soluble carbodiimide reagent. Island structures were observed which were thought to be composed of immobilized catalase molecules. This interpretation was supported by observations of the effect of reversing the polarity of the bias voltage. The application of a platinum/carbon film was found to produce rigidly immobilized protein molecules, and yielded the best resolution. However, covalent coupling techniques offer particular promise as generally applicable methods by which assemblies of proteins may be prepared, facilitating the investigation by scanning tunneling microscopy of their responses to physical and chemical stimuli.

Immobilization of a Monolayer of Bovine Serum Albumin on Gold Nanoparticles for Stereo‐specified Recognition of Dansyl‐norvaline

“…The formation of such monolayer systems is extremely versatile and can provide a method for the in vitro development of bio-surfaces, which are able to mimic molecular recognition processes in nature. 17 To date, molecular interactions occurring at biomolecular monolayer surfaces can be studied with a wide range of analytical techniques such as: Surface Plasmon Resonance, [18][19] Scanning Probe Microscopies, [20][21][22] Reflection Absorption Infrared Spectroscopy, 17,21 Surface Enhanced Raman Spectroscopy, 23 and electrochemical techniques. [24][25][26] However, the use of the well-developed automated high performance liquid chromatography (HPLC) method for this purpose is limited.…”

Layer-by-Layer Method for Immobilization of Protein Molecules on Biochip Surface

“…The surface has been modified by various methods, including Langmuir-Blodgett transfer [11][12][13][14], covalent binding [15][16][17][18], and spontaneous adsorption from solution [19][20][21] J.…”

Molecular Characterization of Beef Liver Catalase by Scanning Tunneling Microscopy

“…However, owing to the grains of Pt-C with diameters of 0.8 to 3.5 nm and height of 0.2 to 1.2 nm, high resolution STM image of this enzyme can not be obtained. Leggett et al made use of thiohydracrylic acid as a couple reaction agent in which one end (thio group) assembled on the Au surface and the other end (carboxyl group) reacted easily with the amino of polypeptide chain to fix catalase molecules on the modified Au surface [32,33]. Nevertheless, thiohydracrylic acid is commonly used as an effective reductive agent to break the disulfide bridge of a protein, and the authors paid little attention to the possible denaturation of the catalase immobilized by that method.…”