Imagine a small integrated biomedical analysis laboratory, connected to your home computer, which would be capable of diagnosing illnesses, a lack of vitamins, or the over-presence of substances from samples of blood, urine or saliva. This hypothetic system would be able to give a diagnosis within minutes, finally advising the user about the optimal targeted medicines to take or the right specialist to consult for fast recovery. Of course this system will not be ready in the near future, but this thesis aims to bring some new elements to this exciting project by investigating the diffusion of proteins in well-defined nanometer-sized confined areas. Understanding molecular dynamics in nanoconfinement volumes is fundamental for designing the appropriate lab-on-a-chip devices able to transport, pre-concentrate, separate and sense biomolecules. However, a multitude of phenomena occurring at the nanoscale are still to be discovered and currently, there is a lack of accurate theoretical models to predict the transport of proteins in nanofluidics. Based on measurements performed in 50 nm high 1D nanochannels, where the surface-to-volume ratio is extremely high, protein-surface interactions were initially investigated. Using electrical measurements, the adsorption and desorption kinetics of highly concentrated bovine serum albumin proteins was characterized in different scenarios. Ionic strength conditions were identified, where the electrical conductance is dominated by volume effects due to the adsorbed or bound proteins, leading to potential applications of rapid immunology on-chip. Other situations, where the protein charges were directly influencing the nanochannel conductance, were also highlighted, giving a better understanding of how the adsorbed proteins counterions modify the surface charges. Furthermore, the transport of single proteins diffusing through nanochannels was analyzed using fluorescence correlation spectroscopy. Direct measurements inside nanochannels has allowed the identification of different regimes of interacting proteins, depending on the thickness of the electrical double layer (constituted of immobile ions which equilibrate the surface charges). Taking into account the steric exclusion due to the small channel size, the reversible surface adsorption, and the exclusion-enrichment effect due to the charge of the proteins and ionic strength of the solution, novel theoretical models describing the hindered diffusion of proteins were elaborated. Conditions where the diffusion of proteins through the nanochannels were of the same magnitude as in the bulk were both predicted and experimentally verified. Finally, a novel method is presented to measure the apparent diffusion coefficients of fluorescently-labeled molecules directly inside a nanofluidic system. This technique, based on steady-state dispersion of proteins in a transversal nanoslit, demonstrates that under specific ionic conditions, the apparent diffusion coefficient of wheat germ agglutinin proteins is four orders of magnitude lower than its free diffusion value. Based on this system, the binding affinity of two different proteins was directly measured, demonstrating the potential of this method to be used as a biosensor for quantifying rapid protein complex formation. This thesis mainly deals with fundamental studies related to surface physics and physical chemistry applied to life sciences. The work points out novel, important, experimentally-verified complements to define solid theoretical models, in order to go forward with the design of complex nanofluidic systems applied to biomedical and biological applications.