## 1 Introduction  
Nanomaterials have emerged as promising agents in biomedicine, with applications of nanoparticles ranging from photodynamic therapy to drug delivery or biological sensing [1]. Based on their dimensionality, nanomaterials have been classified as zero-dimensional nanoparticles, one-dimensional nanomaterials such as nanorods or nanotubes, two-dimensional nanomaterials such as nanosheets or nanofilms, and three-dimensional nanoparticles [2]. Among the broad types of nanoparticles, gold nanoparticles have drawn special attention due to their unique properties, such as controllable size, optical and electronic characteristics, and large surface-to-volume ratio [3; 4]. Moreover, their high surface energy allows them to interact with various biomolecules from biological fluids. The resulting nanoparticles, coated with various proteins (knownas protein corona) gain a new biological identity, as the protein layer can significantly affect their biodistribution and functionality [5; 6]. A protein corona is actually made up of two component layers, the \"hard\" corona and the \"soft\" corona. The \"hard\" corona is represented by strongly-adsorbed proteins at the nanomaterial surface that usually remains irreversibly bounded. In contrast, the \"soft\" corona consists of proteins with a weaker binding affinity, so it exhibits a dynamic exchange [7; 8]. Because of these rearrangements, the surface of a protein corona changes over time, which is reflected in the biological performance of the nanoparticle. The screening of the nanoparticle surface by the corona has proved applicable in active targeting via specific ligands. However, it has been shown that their binding capacity is lost when these functionalized particles are placed in a biological environment [9; 10]. Apparently, this loss of targeting specificity is caused by their interaction with other proteins in the medium and the formation of the protein corona. As a result, when placed in a biological medium, nanoparticles can easily fail to recognize and target relevant pathways. As nanoparticles continue to enter cells, the failure to target the intended pathways may lead to the accumulation in targeted and even in the non-targeted cells [11]. Thus, it is critical to establish analytical methods for the in-depth characterization of the protein corona and its interaction with other proteins. Thus far, the nanoparticle-protein corona complexes have been mainly characterized qualitatively by assessing the factors which control protein corona formation (nanoparticle size, surface charge, morphology, pre-coating, abundance, and association/dissociation rate of proteins) or the conformational changes of proteins. The methods used for the primary characterization of protein corona have been UV-visible spectroscopy, Fourier transformed infrared spectroscopy, dynamic light scattering (DLS), circular dichroism spectroscopy, and isothermal titration calorimetry [12; 13]. All these methods have provided valuable qualitative information about the protein corona, but the available methods for the direct quantification of gold nanoparticles' (AuNPs) protein coronas are scarce, and research on this topic is still emerging. The relative quantification of different proteins forming AuNPs' protein coronas was achieved by [PERSON] et al. by using gel-based densitometry [14]. Furthermore, DLS was employed by [PERSON] et al. for quantification, but the method lacks sensitivity for low amounts of protein [15]. For polymeric nanoparticles, the total quantification of proteins can be relatively easily achieved by using spectroscopic UV-Vis assays (i.e., by Bradford) since there are no interferences. In contrast, the quantification of AuNPs' protein coronas can be tedious since their absorption peak is in the same range as colorimetric protein assays [16] and no standard method has been established.