**1.1. Biomarkers discovery**

A biomarker is defined by the Food and Drug Administration (FDA) as "A characteristic that is objectively measured and evaluated as an indicator of normal biological processes, pathogenic processes, or pharmacological responses to a therapeutic intervention."-

It is possible to distinguished three categories depending on the application of the biomarker: diagnostic biomarkers used for disease detection, prognostic biomarkers used for predict the course of a particular disease such as, recurrence, progression, and survival and predictive biomarkers used for predict the response to treatment that could be subsequently applied in patient assessment [1].

During last years, biomarker research and its translation into the clinics have been accelerated by protein microarrays due to the extraordinary capacity for identifying of valuable biomarkers in a short period of time without the requirement of any prior in-depth knowledge into the mechanism of disease progression.

Moreover, protein microarrays are reliable for analyzing targeted/non-targeted biomarkers presented in mostly of human proximal body fluids (such as plasma, serum, synovial liquid,…)- with a wide dynamic range. In contrast with other proteomic strategies, protein microarrays- avoid the sample pre-fractionation. Thus, for example, serum, plasma, urine and tissue extracts which are complex and non-fractionated proteome mixtures, could be used for experimentation. For this reason, among others, protein microarrays offer a powerful technology for functional proteomics analysis in HT format.

 Microarray technologies, like DNA arrays, printed dense spots of capture ligands immobilized onto a solid support that are exposed to samples containing corresponding binding molecules (often called queries), allowing the simultaneous analysis of thousands of capture targets within the same assay. Ekins and collaborators described these binding events based on miniaturization as the key parameter. They predict that a system that uses small amounts of capture molecules and a small amount of sample could be more sensitive than a system using a hundred times more material. In fact, this is the case when K-<-0.1 (where K is the affinity constant between ligand and target) [9].

The capture ligand is presented in a confined area of array, reducing its diffusion. The specific binding event with its target takes place with the highest signal intensities and optimal signal-to-noise ratio could be achieve in these small spots [9] (**Figure 2**). An immunoassay


**Figure 2.** Process of microarray technology.

in an array format displays sensitivities in the pM to fM range, enabling test low-abundant (pg/mL) analytes in crude proteomes with a small volume of sample. In many cases, protein microarrays show a relevant advantage in clinical applications because the samples to test are minimal [10].

For that reason, protein array technology needs to use a multiplex and highly sensitive protein assay capable of handling and resolving complex proteomes with limited available sample [10].

Recently, several types of protein microarrays have been developed and applied as multiplex throughput assay in several biological characterization. Here, it is described the principal features of protein microarrays.
