Miniaturised and Quantitative Techniques Targeting Prostate Cancer

The prostate-specific antigen (PSA) test used in the clinic today has been subjected to suspicions regarding specificity and sensitivity, since cases of benign prostatic hyperplasia or prostatitis are hard to distinguish from those with prostate cancer. This thesis is based upon five original papers describing efforts to develop antibody microarrays enabling improved diagnostic prostate cancer analysis. All assay developments were performed on our in-house developed and microstructured silicon surfaces, which generate increased spot signals and rapid analysis. On these surfaces, two common techniques, sandwich and reverse phase antibody microarrays were compared from a clinical perspective by analysing PSA. Using FITC-labeled detection we found the sandwich format to outperform the reversed phase assay; the sandwich assay gave five orders of magnitude better limit of detection. In an attempt to also address low abundant markers in blood serum or plasma, signal amplification was developed using europium-incorporated nanoparticles coated by streptavidin. The nanoparticles generated a signal amplification of one order of magnitude as compared to europium-labeled streptavidin. Furthermore, we validated our antibody microarray platform using 80 blood plasma samples from the clinical routine; we developed a true quantitative sandwich antibody microarray for PSA analysis in crude EDTA-plasma samples, and benchmarked the assay to a well-documented DELFIA. The first steps to a duplex assay were also taken by analysing total and free PSA on the same chip. In addition, proof-of-principle for a future lab-on-a-chip approach was shown by combining plasmapheresesis, and subsequent PSA analysis by a sandwich antibody microarray assay. The final work describes a nanovial array for rapid protein digestion and Maldi-TOF analysis; two prostate cancer biomarkers were successfully identified after 30 seconds of tryptic digestion. In conclusion, we have now developed protein chip technology to better suite the clinic within the field of prostate cancer diagnostics