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We have developed the immuno-pillar chip for rapid and easy-to-use immunoassay [1].However,in order to apply clinical diagnosis,it is necessary to improve the performance of the immuno-pillar chip.In conventional immuno-pillar chip,antibodies were immobilized on polystyrene beads by physical absorption.In this work,we modified the immobilization method of the antibody.The antibody was covalently immobilized on the beads by using affinity beads.Using a new immuno-pillar chip,we demonstrated the detection of C-reactive protein (CRP) in serum.A photogragh of the immuno-pillar chip is shown in Fig 1.The mixture of anti-CRP antibodies covalently immobilized beads (5 μm diameter),photoprepolymer (PEG-based polymer) and photo initiator was introduced into the microchannel.UV light (365 nm) was irradiated through a photomask on the microchannel.The photoprepolymer was polymerized only in the exposed areas,and the exposed areas became hydrogel pillars including many anti-CRP immobilized beads.A schematic of the immuno-pillar chip is shown in Fig 2.Assay procedure was described elsewhere [1].The fluorescence signal from the immuno-pillar was detected by an inverted fluorescence microscope equipped with a CCD camera.The fluorescence image and bright field image of the immuno-pillar are shown in Fig 3.First,we assayed standard samples (1% BSA-PBS) spiked with CRP.The calibration curve of CRP is shown in Fig 4 (a).The limit of detection (LOD) was 0.1 ng/mL and linear dynamic range was quite good.The total assay time was 33 min.Second,we assayed human serum samples spiked with CRP.The calibration curve of CRP is shown in Fig 4 (b).The LOD was 10 ng/mL.Like the result of standard samples,the result showed good preformance for the surum samples.We developed a new immuno-pillar chip with affinity beads.Using the chip,we succeeded in high sensitive detection of CRP in serum.The LOD for standard samples was two orders of magnitude lower than that of previous immuno-pillar chip.