ABSTRACT
A colorimetric microarray for the multiplexed detection of recurrence of bladder cancer including protein markers interleukin-8 (IL8), decorin (DCN), and vascular endothelial growth factor (VEGF) was established to enable easy and cheap read-out by a simple office scanner paving the way for quick therapy monitoring at doctors' offices. The chip is based on the principle of a sandwich immunoassay and was optimized prior to multiplexing using IL8 as a model marker. Six different colorimetric assay formats were evaluated using a detection antibody (dAB) labeled with (I) gold (Au) nanoparticles (NPs), (II) carbon NPs, (III) oxidized carbon NPs, and a biotinylated dAB in combination with (IV) neutravidin-carbon, (V) streptavidin (strp)-gold, and (VI) strp-horseradish peroxidase (HRP). Assay Format (III) worked best for NP-based detection and showed a low background while the enzymatic approach, using 3,3',5,5'-tetramethylbenzidine (TMB) substrate, led to the most intense signals with good reproducibility. Both assay formats showed consistent spot morphology as well as detection limits lower than 15 ng/L IL8 and were thus applied for the multiplexed detection of IL8, DCN, and VEGF in synthetic urine. Colorimetric detection in urine (1:3) yields reaction signals and measurement ranges well comparable with detection in the assay buffer, as well as excellent data reproducibility as indicated by the coefficient of variation (CV 5-9%).
Subject(s)
Biosensing Techniques/methods , Colorimetry/methods , Neoplasm Recurrence, Local/diagnosis , Protein Array Analysis/methods , Urinary Bladder Neoplasms/diagnosis , Biomarkers, Tumor/urine , Decorin/urine , Gold/chemistry , Horseradish Peroxidase/metabolism , Humans , Interleukin-8/urine , Limit of Detection , Metal Nanoparticles/chemistry , Neoplasm Recurrence, Local/urine , Urinalysis , Urinary Bladder Neoplasms/urine , Vascular Endothelial Growth Factor A/urineABSTRACT
CONTEXT: About 50-70% of patients with non-muscle invasive bladder cancer (NMIBC) experience relapse of disease. OBJECTIVE: To establish a panel of protein biomarkers incorporated in a multiplexed microarray (BCa chip) and a classifier for diagnosing recurrent NMIBC. MATERIALS AND METHODS: Urine samples from 45 patients were tested. Diagnostic performance was evaluated by receiver operating characteristic (ROC) analysis. RESULTS: A multi biomarker panel (ECadh, IL8, MMP9, EN2, VEGF, past recurrences, BCG therapies and stage at diagnosis) was identified yielding an area under the curve of 0.96. DISCUSSION AND CONCLUSION: This biomarker panel represents a potential diagnostic tool for noninvasive diagnosis of recurrent NMIBC.