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Fast and efficient detection of Cobra cardiotoxin (CTX) protein molecules on biochip surfaces is an example of application in biotechnology. One potential application of mixed self assembled monolayers (SAMs) as chip surfaces yield different binding affinities of the CTX proteins, a series of studies on the interaction force between CTX proteins and the mixed SAMs surfaces formed from mixtures of two thiols with the same/different chain lengths and/or with the same/different terminal groups will be investigated. In these dual papers, the mixed SAMs of n-alkinethiol SAMs of different chain lengths are chosen as the first examples of this series due to the simple functions of the mixed SAMs surface structure. Thus, the adhesion force measurements of CTX protein molecules on mixed SAMs of n-alkinethiol SAMs of different chain lengths: 1-decanethiol (C9) and 1-hexanethiol (C5) with different mixing ratios are developed and conducted using atomic force microscope (AFM). There are two major tasks in Part I of the dual papers: the development of the AFM measurements providing reliable information, and selection of the surface with highest binding affinity among this mixed SAMs group. Results indicate that the adhesion forces for CTX protein molecules on mixed SAMs with mixing ratio (χC9) of 0.25, 0.5, 0.75 and 1, are 1.26, 1.8, 1.38, and 1.25 folds respectively, compared with the adhesion force of CTX protein molecules on the C5 surface only. Therefore, the SAM surfaces of χC9 = 0.5 is the best choice as a biomaterial sensor of this group of mixed SAMs because the strongest binding force and highest efficiency. Effects of the loading force of the AFM operation, the radius of curvature of the AFM tip, and the AFM tip endurance as well as control experiments were examined to ensure the quantitative determination of adhesion force for AFM measurement. The physical mechanism of protein adsorption on SAM surfaces will be studied and analyze- - d by molecular dynamics (MD) simulations and will be reported in Part II of the dual papers to compensate the limited information on the interaction taking place at atomic level that experiments cannot provide.