C-reactive protein and interleukin 6 microfluidic immunoassays with on-chip pre-stored reagents and centrifugo-pneumatic liquid control.

We present a fully automated centrifugal microfluidic method for particle based protein immunoassays. Stick-pack technology is employed for pre-storage and release of liquid reagents. Quantitative layout of centrifugo-pneumatic particle handling, including timed valving, switching and pumping is assisted by network simulations. The automation is exclusively controlled by the spinning frequency and does not require any additional means. New centrifugal microfluidic process chains are developed in order to sequentially supply wash buffer based on frequency dependent stick-pack opening and pneumatic pumping to perform two washing steps from one stored wash buffer; pre-store and re-suspend functionalized microparticles on a disk; and switch between the path of the waste fluid and the path of the substrate reaction product with 100% efficiency. The automated immunoassay concept is composed of on demand ligand binding, two washing steps, the substrate reaction, timed separation of the reaction products, and termination of the substrate reaction. We demonstrated separation of particles from three different liquids with particle loss below 4% and residual liquid remaining within particles below 3%. The automated immunoassay concept was demonstrated by means of detecting C-reactive protein (CRP) in the range of 1-81 ng ml-1 and interleukin 6 (IL-6) in the range of 64-13 500 pg ml-1. The limit of detection and quantification were 1.0 ng ml-1 and 2.1 ng ml-1 for CRP and 64 pg ml-1 and 205 pg ml-1 for IL-6, respectively.