We propose a simple and robust microarray immunoassay

We propose a simple and robust microarray immunoassay platform for simultaneous detection of two prostate cancer related biomarkers (PSA and hK2) in serum. Fig. 1 shows the layout of the platform, which simultaneously incorporates a standard curve and real sample assaying. PDMS wells in a 3×9 format were assembled on epoxy coated slide glass. Epoxysilane based surface chemistry was selected for its high binding capacity and the known low background noise [25,26]. The two first 9 well rows were reserved to assaying a standard curve for PSA and hK2 (standard samples). The last 9 wells were dedicated to immunoassaying PSA and hK2 spiked serum samples (see Fig. 2b). 4×4 antibody arrays were spotted in each well. For best performance of the immunoassay platform, we evaluated three concentrations of the two spotted capture atp citrate lyase inhibitor and measured fluorescent signals of both target proteins, PSA and hK2. By optimization of the capture antibody concentration, an excellent sensitivity of each of the two biomarkers, PSA and hK2, was obtained with a LOD (limit of detection) less than 10pg/mL for both assays. Moreover, a 104–105 order of magnitude dynamic range, depending on the concentration of the capture antibody of PSA and hK2 assay was obtained. The platform allows measuring both standard samples and complex samples in parallel and this makes it possible to reduce analytical error of slide-to-slide variance.
Material and methods
Results and discussion
Conclusion In this work, we developed a duplex microarray immunoassay platform for PSA and hK2. By optimization of the concentration of the spotted capture antibody, the platform displayed an LOD of 5pg/mL in spiked serum samples for both target proteins without any significant cross-reaction or interference from unspecific binding. Our assay platform has comparable sensitivity to other ultra-sensitive detection methods such as nano-particle based detection method [29,30], single molecular digital ELISA systems [31,32], 3-D porous silicon assay platforms [33,34] for PSA detection and highly optimized ELISA system for hK2 detection [35,36]. Moreover, this platform showed selectivity to the target biomarker as compared to the counter antigen, PSA and hK2 respectively. The optimization procedure described herein enables the development of multiplex antibody microarrays for simultaneous measurement of several biomarkers related to prostate cancer. As an initial step towards a truly multiplex assay we realized an optimized duplex assay of both PSA and hK2 spiked in serum. Several other proteins in the human kallikrein family have strong correlations with malignant prostatic disease [37,38] and are thus in focus accomplish a multiplex detection of these biomarkers and potentially provide improved prostate cancer diagnostics.
Conflict of interest
Acknowledgement This study was partly supported by RIKEN FPR program and JSPS KAKENHI Grant Number 25350581. This project is also supported by Korea Ministry of Environment as “EI project” (ERL E211-41003-0007-0).
Introduction A high percentage of the world’s population lives in areas with high risks to water security [1]. Climate change, rapid urbanization, increasing population and extensive agriculture all act as global threats to the Earth’s supply of fresh water. Clean and reliable drinking water can be ensured through periodic and extensive testing at key points of the water infrastructure [2]. To enable this, toxicity tests need to be rapid, accurate, portable and low cost to provide affordable water security especially in developing countries [3]. Current methods need long processing times for analysis of water composition to provide precise information of the chemical substances present in the water [4]. An alternative to this method is cell-based sensors which can detect the presence of toxicants by monitoring the cell viability. Cell-based sensors are highly attractive for detection of chemical, biological and environmental toxicants, because living cells are sensitive environmental toxicants [5,6]. Sensitivities of cell-based sensors to biological toxins and chemical reagents depend on several factors such as: the type of cells, the properties of the agents and the sensing technique. Cell-based biosensors can employ fluorescence probes [4], or gravimetric and impedance techniques [7] to detect the presence of toxicants.