Laser-assisted protein micropatterning in a thermoplastic device for multiplexed prostate cancer biomarker detection

Immunoassays are frequently used for analysis of protein biomarkers. The specificity of antibodies enables parallel analysis of several target proteins, at the same time. However, the implementation of such multiplexed assays into cost-efficient and mass-producible thermoplastic microfluidic platforms remains difficult due to the lack of suitable immobilization strategies for different capture antibodies. Here, we introduce and characterize a method to functionalize the surfaces of microfluidic devices manufactured in the thermoplastic material cyclic olefin copolymer (COC) by a rapid prototyping process. A laser-induced immobilization process enables the surface patterning of anchor biomolecules at a spatial resolution of 5 μm. We employ the method for the analysis of prostate cancer associated biomarkers by competitive immunoassays in a microchannel with a total volume of 320 nL, and successfully detected the proteins PSA, CRP, CEA and IGF-1 at clinically relevant concentrations. Finally, we also demonstrate the simultaneous analysis of three markers spiked into undiluted human plasma. In conclusion, this method opens the way to transfer multiplexed immunoassays into mass-producible microfluidic platforms that are suitable for point of care applications.