br Conflict of interest br Acknowledgements

Conflict of interest
Acknowledgements Authors would like to thank the financial support of the European Community’s Seventh Framework Programme FP7-SME-2013 “DEMOTOX” – Proposal no 604752, the Center for Life Nano Science@Sapienza, Istituto Italiano di Tecnologia (Rome, Italy) and the Italian Ministry of Education, University and Research (MIUR) through PRIN2010-2011 project “ARTEMIDE” and the University Project Research 2013 – prot. C26A13HKFB “Lab-on-Chip system for mycotoxin detection in food commodities”.
Introduction Increasing levels of PSA in the blood circulation can be caused by prostate cancer (PCa), but it also appears at benign prostate diseases since elevated PSA is specific to prostate disorders and not only to PCa. In male blood, PSA levels vary from <0.1ng/mL to 10μg/mL [1], though elevated levels above 4ng/mL recommend further diagnostic steps to be taken including prostate biopsy. Studies of larger cohorts show that men with PSA above this threshold had cancer detection rates of 27–44% [2,3]. On the other hand, there are several studies reporting that prostate cancer were detected in 6.6% of the men with a PSA level less than 0.5ng/mL PSA and in 27% of the men with PSA ranging from 3.1ng/mL to 4ng/mL [4,5]. PSA screening tests are reported to have a significant correlation with a 20% reduction in cancer mortality, however it has drawbacks such as leading to unnecessary and painful biopsies due to the low specificity of the PSA assay [6,7]. Therefore, requirement for additional prostatic tumor markers are increasingly high. Human glandular kallikrein 2 (hK2) is a serine protease having structurally similarity with PSA [8,9] and monomeric polypeptides of 237 amino atp citrate lyase residues having around 80% sequential homology to PSA [10]. hK2 has been considered as another PCa biomarker since the hK2 levels are increased in serum from PCa patients relative to individuals with benign prostate hyperplasia (BPH) [11,12]. Moreover, hK2 is known to act as an independent predictor of PCa diagnosis in case of mildly elevated PSA level between 4ng/mL and 10ng/mL so called “gray zone” [13,14]. Concentration of hK2 in human prostate is approximately 10–50% of the PSA level and it is 50- to 100-fold lower than that of the PSA concentration in human blood [15,16]. Considering PSA’s cut-off range (4ng/mL), the hK2 assay should need more sensitive detection with a limit of detection (LOD) of 40pg/mL or lower. Antibody microarrays are known to have potential for high throughput multiplex screening of biomarkers and is also proposed as a tool for the detection of low abundant protein biomarkers in human biofluids such as serum, urine and cerebrospinal fluid (CSF) [17]. The microarray format can ultimately offer advantages in terms of a low amount of antibody consumption, highly sensitive readout, and multiplex performance. Such developments hold promise of earlier diagnosis of disease, reducing the need for biopsy and providing post therapy monitoring of patients for recurrence [18,19]. Generally, there are two types of immunoassays, sandwich (forward phase) immunoassay and reverse phase assay [20–22]. Sandwich immunoassays have the advantages of sensitive and selective detection of antigen since two antibodies are used for specifically captured and enrichment of the antigen and followed by specific detection of the captured species. It is also a suitable method for the detection of antigens in complex samples since crude biofluids can be analyzed with no minimal sample pretreatment before performing the assay [23]. To improving the detection sensitivity of the sandwich type immunoassay, optimizations of the capture antibody such as affinity or/and surface density are one of most important steps in microarray format [24] since a major hurdle in sensitive assaying is the elimination of antigen cross-reaction and unspecific binding to antibodies.