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%).
Highlights
Bladder cancer (BCa) is a serious malignancy of the urinary tract and prominent for its high rate of recurrence concerning 50% of all treated patients
A number of urinary biomarkers (including fluorescence in-situ hybridization (FISH), ImmunoCyt, and nuclear matrix protein 22 (NMP22)) and investigational urine markers [2] exist, but none of these markers has yet been shown to decrease the need for cystoscopy
Biomarkers identifying patients most likely to respond to chemotherapy for example would have enormous utility, reducing morbidity and improving quality of life and significant cost savings to health providers
Summary
Bladder cancer (BCa) is a serious malignancy of the urinary tract and prominent for its high rate of recurrence concerning 50% of all treated patients. To intervene recurrence of BCa, routine cytology and cystoscopy are done, representing the gold standard. These methods are expensive, time-consuming, and invasive and lead to urinary infections in up to 16% of patients. A number of urinary biomarkers (including fluorescence in-situ hybridization (FISH), ImmunoCyt, and nuclear matrix protein 22 (NMP22)) and investigational urine markers [2] exist, but none of these markers has yet been shown to decrease the need for cystoscopy. In previous work [3], we compiled a list of relevant bladder cancer protein markers based on extensive literature search (>1000 research articles) and prior defined inclusion and exclusion criteria. Microarray technology, a miniaturized high throughput analysis method, that can measure a high number of biomarkers in parallel was applied as a screening tool to narrow down the biomarker candidates and to evaluate the marker profiles in a clinical
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